Structural and mutagenesis studies evince the role of the extended

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Structural and mutagenesis studies evince the role of the extended protuberant domain of ribosomal protein uL10 in protein translation Kwok-Ho Andrew Choi, Lei Yang, Ka-Ming Lee, Conny Wing-Heng Yu, David Karl Banfield, Kosuke Ito, Toshio Uchiumi, and Kam-Bo Wong Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00528 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1

Structural and mutagenesis studies evince the role of the

2

extended protuberant domain of ribosomal protein uL10 in

3

protein translation

4 5

Kwok-Ho Andrew Choi†, Lei Yang†, Ka-Ming Lee†, Conny Wing-Heng Yu†, David K.

6

Banfield§, Kosuke Ito‡, Toshio Uchiumi‡ and Kam-Bo Wong*,†

7 8

† School

of Life Sciences, Centre for Protein Science and Crystallography, State Key

9

Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, Hong

10

Kong, China

11

§ Division

of Life Science, Hong Kong University of Science and Technology, Clear Water

12 13 14

Bay, Hong Kong, China ‡ Department

of Biology, Faculty of Science, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2191, Japan

15

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16 17


Page 2 of 46

Page 3 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

18

ABSTRACT

19

The lateral stalk of ribosomes constituted the GTPase-associated centre and is responsible for

20

recruiting translation factors to the ribosomes. Eukaryotic stalk contains a P-complex, in which

21

one molecule of uL10 (formerly known as P0) protein binds two copies of P1/P2 heterodimers.

22

Unlike bacterial uL10, eukaryotic uL10 has an extended protuberant (uL10ext) domain inserted in

23

the N-terminal RNA-binding domain. Here, we determined the solution structure of the extended

24

protuberant domain of Bombyx mori uL10 by nuclear magnetic resonance spectroscopy.

25

Comparison of the structures of the B. mori uL10ext domain with eRF1-bound and eEF2-bound

26

ribosomes revealed significant structural rearrangement in a “hinge” region surrounding Phe183,

27

a residue conserved in eukaryotic but not in archaeal uL10. 15N-relaxation analyses showed that

28

residues in the hinge region have significant large values of transverse relaxation rates. To test the

29

role of the conserved phenylalanine residue, we created a yeast mutant strain expressing a F181A

30

variant of uL10. In-vitro translation assay showed that the alanine substitution increased the poly-

31

phenylalanine synthesis by ~33%. Taken together, our results suggest that the hinge motion of the

32

uL10ext domain facilitates the binding of different translation factors to the GTPase-associated

33

centre during protein synthesis.

34


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

35

Page 4 of 46

INTRODUCTION

36

The lateral ribosomal stalk of the large subunit of ribosomes constitutes the GTPase-associated

37

center and is responsible for the recruitment and function of translation factors 1–3. Ribosomal stalk

38

in bacteria, archaea and eukaryotes all contains an anchorage protein, uL10 (formerly L10 in

39

bacteria; P0 in archaea and eukaryotes) that binds the stalk to the 23S/28S rRNA via an N-terminal

40

RNA-binding domain that is homologous in bacteria, archaea and eukaryotes. While the C-

41

terminal domains of archaeal and eukaryotic uL10 are structurally distinct from bacterial uL10 4,5,

42

they are functionally analogous in binding multiple copies of small stalk proteins (bL12 in bacteria;

43

P1 in archaea; P1/P2 in eukaryotes). Bacterial stalk is consisted of uL10 forming a complex with

44

2 to 3 copies of bL12 homodimers

45

copies of P1 dimers 8, while eukaryotic uL10 forms a complex with 2 copies of P1/P2 heterodimers

46

in eukaryotic ribosomes

47

P1/P2 heterodimers and characterized its dynamics behavior by NMR spectroscopy

48

showed that P1 and P2 contain an N-terminal dimerization domain and a flexible C-terminal tail

49

that are responsible for fetching the translation factors and ribosome inactivating proteins to the

50

ribosomes 4,5,14–20. In Saccharomyces cerevisiae, there were two isoforms (α and β) of P1 and P2

51

and they form P1α/P2β and P1β/P2α heterodimers, and the assembly and architecture of how the

52

heterodimers interact with uL10 were studied using small-angle X-ray scattering and mutagenesis

53

studies13,21,22. It has been shown that multiple copies of P1/P2 heterodimers on the ribosomal stalk

54

play an important role in increasing the fidelity of protein translation23.

4,9–13.

6,7.

In archaeal stalk, archaeal uL10 forms a complex with 3

We have previously determined the solution structure of human 4,11,12

, and

55 56 57

Archaeal and eukaryotic uL10 also differ from bacterial uL10 in containing an extended protuberant (uL10ext) domain inserted in the N-terminal RNA-binding domain


8,24,25

(Figure 1).

Page 5 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

58

There are several pieces of evidence that suggest the function of the uL10ext domain in protein

59

translation. First, truncation of the uL10ext domain in Saccharomyces cerevisiae uL10 reduced

60

the growth rate and the amount of eukaryotic elongation factor 2 (eEF2) bound to the ribosome26.

61

Second, mutations in the uL10ext domain confer resistance to sordarin, an anti-fungal agent that

62

stabilizes the eEF2/ribosome complex27–29. Third, we have previously shown that truncation of the

63

uL10ext domain in Pyrococcus horikoshii and Bombyx mori uL10 decreased the eEF2-dependent

64

GTPase activity and polyphenylalanine synthesis in hybrid ribosomes reconstituted with E. coli

65

ribosome cores and archaeal/eukaryotic stalk complexes25,30. Recently, the domain is also found

66

to be interacting with general control nonderepressible 2 (GCN2) that phosphorylates eukaryotic

67

initiation factor 2α in response to stress 31

68 69

Crystal structure of an archaeal uL10 from Methanococcus jannaschii was determined

70

previously – the uL10ext domain forms a distinct protuberant domain connecting to the RNA-

71

binding domain via connecting loops32. However, in the eukaryotic 80S ribosome structures

72

determined by X-ray crystallography and cryo-electron microscopy, the uL10ext domain was often

73

not modelled due to the absence of interpretable densities there. Even in cases where the uL10ext

74

domain was modelled, its densities were less well-defined compared to other regions of the

75

ribosomes. These observations suggest that the uL10ext domain is structurally flexible. In this

76

study, we determined the solution structure of the extended protuberant domain of Bomyx mori

77

uL10 (BmuL10ext) and characterized its backbone dynamics by NMR spectroscopy. Significant

78

high values of transverse relaxation rates were observed for residues in a “hinge” region of the

79

uL10ext domain (consisting of Phe183, a residue conserved in eukaryotic but not in archaeal uL10,

80

and the surrounding residues in 1/ 2 and 5/ 6). Interestingly, significant structural


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

81

rearrangements were observed when comparing the solution structure of uL10ext to that in eRF1-

82

bound and eEF2-bound ribosomes. Finally, we created a yeast mutant strain expressing a F181A

83

variant (the corresponding residue of Phe183 in yeast uL10 is Phe181) of uL10 and showed that

84

80S ribosomes with the uL10-F181A exhibited increased protein translation. The potential

85

biological role of the uL10ext domain was discussed.


Page 6 of 46

Page 7 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

87 88

Figure 1. Domain organization of eukaryotic uL10 is different from bacterial uL10.

89

All uL10 in eukaryotic, archaeal, and bacterial ribosomes contain a homologous RNA

90

binding domain responsible for anchoring the stalk proteins to the rRNA. In both archaeal

91

and eukaryotic uL10 (formerly known as P0), there is an extended protuberant domain

92

(uL10ext) inserted inside the RNA-binding domain. The C-terminal domain of eukaryotic

93

uL10 contains spine helices that bind two copies of P1/P2 heterodimers, and a conserved

94

motif SDxDMGFxLFx responsible for binding translation factors and ribosome inactivating

95

proteins to the ribosomes. Archaeal uL10 binds two to three copies of P1 homodimers

96

whose C-terminus shares the xGFxALFx sequence for binding translation factors. On the

97

other hand, bacterial uL10 binds two to three copies of bL12 homodimers and lacks the

98

extended protuberant domain and the C-terminal tail. Instead, bL12, which is structurally

99

distinct from P1 or P2, has a C-terminal domain responsible for binding translation factors.

100

The model for eukaryotic stalk is adopted from Lee et al.4. The model for bacterial stalk is

101

adopted from 1ZAV 7. The model for archaeal stalk is generated from Modeller

102

3JSY 32 and 3A1Y 30 as templates.


33

using

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

103 104


Page 8 of 46

Page 9 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

105

MATERIALS AND METHODS

106

Plasmid construction

107

For Escherichia coli expression

108

DNA sequence encoding residue 105-186 of Bomyx mori uL10 (BmuL10ext) was cloned into

109

vector pET-15b (Novagen) and a homemade vector pET151 with a His-GFP tag which was

110

modified from pET-3d (Novagen). The resulting constructs contained either an N-terminal 6xHis-

111

tag followed by a thrombin cleavage site or an N-terminal 6xHis-tag followed by a GFP and a

112

thrombin cutting site.

113

For Saccharomyces cerevisiae expression

114

To construct the balancing plasmid, a PCR fragment containing the DNA sequence of

115

Saccharomyces cerevisiae uL10 (RPP0) with a C-terminal c-Myc tag flanked by alcohol

116

dehydrogenase 1 (ADH1) promoter and ADH1 terminator, was generated by overlap extension

117

polymerase chain reaction and cloned into pRS416 with BamHI site and XhoI site. DNA sequence

118

of RPP0 was amplified from yeast genomic DNA. DNA sequences for ADH1 promoter and ADH1

119

terminator were amplified from pGADT7 (TaKaRa). For constructing the pRPP0-T7, the plasmid

120

remaining in the RPP0-knockout strain after 5’fluoroorotic acid (5’FOA) counterselection, PCR

121

fragment which had the c-Myc tag replaced by a T7-tag was cloned into pRS415 with the same

122

restriction sites. Plasmids containing mutant RPP0 were constructed by site-directed mutagenesis

123

on the coding sequence in the PCR fragment with T7-tag before cloning into pRS415 plasmid. The

124

plasmids used are listed in Table 1.

125 126


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

127

Page 10 of 46

Table 1. List of plasmids for Saccharomyces cerevisiae Plasmid

Description

Source

pRS416

CEN, URA3

(Sikorski & Hieter, 1989) 34

pRS415

CEN, LEU2

(Sikorski & Hieter, 1989) 34

pRPP0-myc

pRS416 carrying RPP0 gene with a c-terminal c-myc This study tag under the control of ADH1 promoter and terminator

pRPP0-T7

pRS415 carrying RPP0 gene with a c-terminal T7 tag This study under the control of ADH1 promoter and terminator

pRPP0-T7-G112A

G112A substitution was made by site directed This study mutagenesis within RPP0 coding sequence in pRPP0T7

pRPP0-T7-A115P

A115P substitution was made by site directed This study mutagenesis within RPP0 coding sequence in pRPP0T7

pRPP0-T7-F181A

F181A substitution was made by site directed This study mutagenesis within RPP0 coding sequence in pRPP0T7

pRPP0-T7-S182P

S182P substitution was made by site directed This study mutagenesis within RPP0 coding sequence in pRPP0T7

128 129

NMR sample preparations

130

Escherichia coli strain Rosetta (DE3) pLysS (Novagen) was transformed with the pET-15b

131

HisBmP0ext or pET-151 His-GFP-BmP0ext plasmids. For unlabeled samples, the transformed

132

strain was cultured in rich medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 5 g/L NaCl, 20

133

g/L tryptone, 5 g/L yeast extract) containing 100 g/ml ampicillin. For labeled samples, the

134

transformed strain was first cultured in rich medium until OD reached ~0.8. The cells were

135

collected and re-suspended in M9 medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 2 mM


Page 11 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

136

MgSO4) containing 2 g/L 13C glucose, 1 g/L 15N ammonium chloride and 100 g/ml ampicillin.

137

The culture was then incubated at 25°C for 2 h and induced for 16 h at 25°C with 1 mM IPTG 35.

138

Cell pellet was resuspended with buffer A (30 ml of 20 mM HEPES, 1 M NaCl, 20 mM Imidazole,

139

pH 7.4) and lysed by sonication. The filtered supernatant of the cell lysate was loaded to a 5 ml

140

HiTrap metal-chelating column (GE Healthcare) preloaded with Ni2+ ions. After extensive

141

washing with buffer A, His-tagged-BmuL10ext was eluted with 300 mM imidazole in buffer A.

142

The fusion tag was cleaved off by thrombin according to the manufacturer’s instructions (GE

143

healthcare) and removed by loading the protein sample to a 5 ml HiTrap metal-chelating column.

144

The flow-through fractions were collected, concentrated to 5 ml and loaded to a HiLoad Superdex

145

75 26/600 gel filtration column (GE Healthcare) pre-equilibrated with 20 mM sodium phosphate,

146

0.15M NaCl, 5% glycerol, pH 7.4. The elution volume of uL10ext was ~240 ml. The protein

147

samples were concentrated to 0.3-1 mM for nuclear magnetic resonance (NMR) experiments.

148 149

Structure determination

150

NMR spectra were collected at 298K using a Bruker Avance 700 MHz spectrometers. Sequential

151

assignment of backbone resonances was obtained by C and C connectivities generated by

152

HNCACB

153

TOCSY-HSQC39,40, H(CC)CONH41, HCCH-TOCSY

154

Stereospecific assignments for the methyl groups of valine and leucine were obtained using a 10%

155

13C-labeled

156

experiments such as 1H,15N-NOESY-HSQC40,46, 1H,13C-NOESY-HSQC

157

NOESY-HSQC

158

dimethyl-4-silapentane-1-sulfonate. All multidimensional NMR data were processed with the

36,37

and CBCA(CO)NH

sample

48,

45.

38

experiments. Side-chain resonances were obtained from 42,43

and 2D 1H-13C HSQC

44

experiments.

Inter-proton distance restraints were obtained from NOESY-type 47, 13C,13C-HSQC-

2D 1H-1H-NOESY49. Chemical shifts were referenced with respect to 4,4-


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 46

159

program TOPSPIN (Bruker Biospin) and analyzed using the program NMRView

160

angle restraints were derived from the TALOS program 51. Hydrogen bond restraints were derived

161

from hydrogen/deuterium-exchange experiments

162

structure elements. Structural calculation was performed using ARIA 2.2 53 and CNS 1.2 54,55 with

163

an initial set of manually assigned distance restraints. The structures were converged in the ﬁrst

164

round of calculation. ARIA-assigned distance restraints were checked manually and were included

165

in subsequent rounds of calculation iteratively. Finally, 10 structures with the lowest total energy

166

and no violation of experimental restraints were selected and deposited as in the Protein Data Bank.

52

50.

Dihedral

and were only included for the secondary

167 168

15N

Relaxation Experiments

169

15N

labeled samples of BmuL10ext were used to determine the 15N longitudinal relaxation rates

170

R1, transverse relaxation rates R2 and heteronuclear NOE using Bruker Avance 700 MHz

171

spectrometers at 298 K. Relaxation delays for measuring R1 were 0.011, 0.128, 0.267, 0.533,

172

0.800, 1.120, 1.440, 1.867 and 3.000 s, and for measuring R2 were 0.017, 0.034, 0.051, 0.068,

173

0.102, 0.136, 0.204, 0.288 and 0.390 s. R1 and R2 rates were obtained by fitting peak intensities to

174

an exponential decay using the program NMRView 50. The standard deviation for the R1 and R2

175

relaxation rates were obtained by Monte-Carlo simulation implemented in NMRView. The steady-

176

state heteronuclear 1H-15N NOE were measured in spectra acquired with and without 1H pre-

177

saturation (with of a series of high-power 120° pulses of 18 µs each) in an interleaved manner 56,57

178

and were defined using the equation (I-Io)/Io, where I and Io are peak intensities measured with or

179

without presaturation, respectively. For all relaxation experiments, a recycle delay of 5 s was used

180

between transients. The NMR spectra for relaxation experiments were processed with NMRPipe


Page 13 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

181

58.

182

spectra.

Uncertainities in intensity measurement were estimated from the root mean square noise of the

183 184

Creation of yeast mutant strains

185

All yeast strains were grown at 30°C. Yeast strains were propagated in either yeast extract

186

adenine dextrose (YPAD) medium or synthetic dextrose (SD) media lacking uracil or leucine.

187

Transformation of plasmids were performed using the lithium acetate/dimethylsulphoxide

188

(DMSO) method 59. The yeast strains used in this study is listed in Table 2.

189

Table 2. List of Saccharomyces cerevisiae strains used in this study Strain

Description

Source

BY4741

MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

(Brachmann et al., 1998) 60

KBP01

BY4741 transformed with pRPP0-myc [CEN URA3 This study PADH1 RPP0-myc]

KBP02

MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rpp0::natRMX4, This study pRPP0-myc [CEN URA3 PADH1 RPP0-myc]

KBP03-WT

MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rpp0::natRMX4, This study pRPP0-myc [CEN URA3 PADH1 RPP0-myc], pRPP0-T7 [CEN LEU2 PADH1 RPP0-T7]

KBP04-WT

MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rpp0::natRMX4, This study pRPP0-T7 [CEN LEU2 PADH1 RPP0-T7]

KBP03-F181A

MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rpp0::natRMX4, This study pRPP0-myc [CEN URA3 PADH1 RPP0-myc], pRPP0-T7F181A [CEN LEU2 PADH1 RPP0(F181A)-T7]

KBP04-F181A

MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rpp0::natRMX4 This study pRPP0-T7-F181A [CEN LEU2 PADH1 RPP0(F181A)-T7]

190 191

RPP0 gene was replaced by natRMX4 selection marker gene by homologous recombination

192

using micro-homology PCR mediated targeting technique 61. PCR fragment was amplified from


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

193

p4339 plasmid containing the 45 bp from both 5’ and 3’ untranslated regions (UTR) immediately

194

outside the RPP0 gene added to a disruption cassette which is composed of natMX6 selection

195

marker gene flanked by TEF1 promoter and TEF1 terminator. The PCR fragment was transformed

196

into mid log-phase yeast culture using lithium acetate/single-stranded carrier DNA/polyethylene

197

glycol method 62. The transformed cells were plated on SD –ura/-leu agar plate with 100 µg/ml

198

nourseothricin (GOLDBIO) and incubated at 30°C for 3-5 days. Successful transformants were

199

tested by PCR for the replacement of the genomic copy of RPP0 with the disruption cassette. The

200

results were verified by DNA sequencing and western blot.

201 202

The RPP0-knockout strain, refer as KBP02 hereafter, was transformed with pRPP0-T7 or

203

pRPP0-T7-F181A plasmids to yield strains (KBP03-WT and KBP03-F181A) that carried two

204

plasmids. The counter-selection was carried out using 5’fluroorotic acid (5’FOA) 63. The KBP03

205

strains were first cultured in 2 ml SD -ura/-leu medium with 100 µg/ml nourseothricin to reach

206

OD600 ~3. Cells were collected, diluted 10-fold and re-suspended in 2 ml SD -leu medium with

207

0.5 mg/ml 5’FOA (GOLDBIO) and 100 µg/ml nourseothricin. The cultures were incubated at

208

30°C. After two days, the cultures were diluted 100-fold in fresh SD -leu medium with 0.5 mg/ml

209

5’FOA and 100 µg/ml nourseothricin and further incubated for 1 day to ensure complete loss of

210

URA3-plasmid. Finally cells were streaked on SD -leu agar plate with 100 µg/ml nourseothricin.

211

The extrusion of pRPP0-myc plasmid was checked by western blot analyses on the strains yielded.

212 213

Purification of ribosomes

214

1 L of yeast culture was grown to OD600 of 0.6 – 1.0 and collected by centrifugation at 10000

215

g, 4°C for 10 min and washed with buffer A (20 mM HEPES, 100 mM potassium acetate, 2 mM


Page 14 of 46

Page 15 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

216

magnesium acetate, 5 mM DTT, pH 7.4). Cell pellet was lysed by a low temperature ultra-high

217

pressure continuous flow cell disrupter (JN-Mini, JNBIO) at 1800 bar, 4°C. The crude lysates were

218

centrifuged at 10,000 g, 4°C for 30 min twice to remove cell debris, yielding the supernatant

219

fraction S30. S30 fraction was submitted to high-speed centrifugation at 184000 g, 4°C for 4 h in

220

a Type 70 Ti rotor (Beckman Coulter). The supernatant was then subjected to 30-70% ammonium

221

sulfate precipitation to obtain the soluble fraction S100, which served as the source of supernatant

222

factors in the in-vitro translation system. The pellet was re-suspended with buffer B (20 mM

223

HEPES, 100 mM potassium acetate, 12 magnesium acetate, 5 mM DTT, pH 7.4) and first

224

centrifuged through a sucrose cushion (33%) in buffer B at 184000 g, 4°C for 4 h in a Type 70 Ti

225

rotor (Beckman Coulter), then through a 10–40% linear sucrose gradient in buffer B at 100,000 g,

226

4 °C for 4 h in a SW32 Ti rotor (Beckman Coulter). Fractions which has a ratio of A260/A280=2:1

227

corresponding to the 80S ribosome-fractions were collected, and finally submitted to

228

centrifugation at 184000 g, 4°C for 4 h in a Type 70 Ti rotor (Beckman Coulter) to collect the

229

ribosomes. The pelleted 80S ribosomes were dissolved in buffer B. The final concentration was

230

determined by A260 as described 64.

231 232

Poly-phenylalanine synthesis assay

233

25 μl reaction mixture containing 80S ribosomes (final concentration= 0.136 μM) and S100

234

(final concentration = 0.72 A280 units), 20 mM HEPES-KOH (pH 7.5), 100 mM potassium

235

acetate, 12 mM magnesium acetate, 0.05 mM spermine (Sigma-Aldrich), 7.5 mM creatine

236

phosphate (Sigma-Aldrich), 1.25 mg creatine kinase (Sigma-Aldrich), 0.1 mM GTP, 5 mg poly(U)

237

(Santa Cruz Biotechnology), 12.5 U of RNAsin plus (Promega) and 12.5 U of micrococcal

238

nuclease (New England Biolabs) were incubated for 30 min at 30 °C. Reactions were terminated


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

239

by adding 975 μl of cold 25% trichloroacetic acid. After the addition of trichloroacetic acid, the

240

samples were further incubated on ice for 1 h. The precipitate was collected on glass filter

241

membranes (Whatman, GF/B), and the hot count value of incorporated [3H]-Phenylalanine was

242

quantified using a liquid scintillation counter. Statistical analysis was performed using the software

243

PRISM (GraphPad).

244 245


Page 16 of 46

Page 17 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

246

RESULTS

247

Structure determination of BmuL10ext

248

The resonance of BmuL10ext was assigned using the triple resonance experiment approach, and

249

the assigned 1H-15N HSQC spectrum is shown in Figure S1. Structure calculations were performed

250

with 1670 interproton restraints, 20 hydrogen bond restraints, and 32 dihedral angle restraints

251

(Table 3). Out of the calculated structures, a set of 10 structures with distance restraint violations

252

less than 0.3 Å and dihedral angle restraint violations less than 5° were selected (Figure 2A).

253

Statistics of the ten structures are summarized in Table 3. The structure of the uL10ext domain is

254

consisted of three pairs of β-strands (  ) and two helices. The two helices pack

255

against each other and the three β-sheets wrap around the two helices (Figure 2B). The topology

256

of BmuL10ext is similar to that found in archaeal uL10 32. Residues in both N- and C-termini are

257

unstructured while residues 108-184 are well defined, with average values for pairwise root-mean-

258

square deviation of 0.50 Å for backbone atoms and 1.04 Å for heavy atoms (Figure 2C).

259 260 261 262


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

263 264 265

Table 3. NMR and refinement statistics for the 10 best structures of BmuL10ext with no restraint violation

266 267

aValues

of mean and standard deviation were reported.


Page 18 of 46

Page 19 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

268 269

Figure 2. Solution structure of the extended protuberant domain (residues 105-186)

270

of B. mori ribosomal stalk protein uL10 (BmuL10ext). (A) Stereodiagram of an

271

ensemble of 10 best structures with the lowest energy and no restraint violations. (B)

272

Topology of BmuL10ext. The extension domain is formed by three pairs of anti-parallel

273

β–sheets that wrap around two helices. (C) Backbone root-mean-square-displacement

274

from the mean structure (RMSD) along the primary sequence. Except for those several

275

residues in both termini, the BmuL10ext domain is well defined.

276


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

277 278

Structural comparison revealed significant structural changes in a hinge region of the P0ext domain

279

In many 80S eukaryotic ribosome structures deposited in the Protein Data Bank, the uL10ext

280

domain was not modelled, possibly due to the dynamic nature of the stalk. Even if the uL10ext

281

domain was modelled (e.g. PDB accession code: 5LZS), the density around the domain was

282

sometimes poor and make structural comparison not reliable (Figure S2). Nonetheless, the

283

eRF1/ABCE1(eukaryotic release factor 1/ATP-binding cassette sub-family E member 1)-bound

284

structures solved by Shao and co-workers (PDB accession code: 5LZV) 65, and the eEF2-bound

285

structure solved by Voorhees and co-workers (PDB accession code: 3J7P) 66 have good densities

286

for the uL10ext domain (Figure S2). We, therefore, compared our structure of BmuL10ext to these

287

structures. The most significant differences were identified in a “hinge” region around a conserved

288

Phe183 residue. In the solution structure of BmP0ext, residues around 1/ 2 (Ala111, Ala115,

289

P118) and 6 (Leu161, Val167) are packed against Phe183 (Figure 3A). In the eRF1/ABCE1-

290

bound ribosome structure, the conserved Phe residue swings towards the loop between 1/2,

291

causing residues around 1/2 and 5/6 to move away from the loop connecting to the RNA-

292

binding domain of uL10 (Figure 3A).

293

Noteworthy, similar structural differences were observed in the uL10ext domain of eEF2-bound

294

and eRF1/ABCE1-bound ribosomes (Figure 3A). We superimposed the structure of uL10 in the

295

eRF1/ABCE1-bound ribosomes (green, Figure S3A) with that in the eEF2-bound ribosomes (red,

296

Figure S3A). Induced by binding of different translation factors, structural changes around the

297

Phe183 residues resulted in a hinge motion that causes the uL10ext domain to undergo a rigid-

298

body movement as shown in Figure S3A.

299


Page 20 of 46

Page 21 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

300

301 302

Figure 3. Structural comparison of the uL10ext domain. (A) Structural differences were

303

observed between the solution structure of BmuL10ext (yellow) and the uL10ext structure

304

from the eRF1/ABCE1-bound ribosomes (PDB: 5LZV

305

conserved Phe183 was found to swing towards the loop between β1 and β2 in the

306

eRF1/ABCE1-bound ribosome, pushing the residues around β1/ β2 and β5/ β6 to move

307

away. Similar structural differences were observed between eEF2-bound (red; PDB: 3J7P

308

66)

309

were coloured in cyan (refer to Figure 4) (B) Sequence alignment of the eukaryotic P0ext

310

domain. Residues around β1/β2 and β5/β6 in the “hinge region” (A111, A115, P118, L161

311

and V167) that form hydrophobic interactions with Phe183 are indicated by triangles.

65).

As indicated by arrows, the

and eRF1/ABCE1-bound ribosomes. L161 and V167 with significant high values of R2


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

312

Residues with significant high values of R2 (L119, L161, V167 and G168, refer to Figure 4)

313

are indicated by circles. Helices and strands are marked as cylinders and arrows,

314

respectively. Residue numbers of BmuL10ext are indicated.


Page 22 of 46

Page 23 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

315 316 317

Biochemistry

15N

relaxation analyses showed significant higher values of transverse relaxation rates in

residues in the hinge region The backbone dynamics of BmuL10ext were characterized by the

15N

longitudinal (R1) and

318

transverse (R2) relaxation rates and 1H,15N-heteronuclear nuclear overhauser enhancement

319

(1H,15N-NOE) (Figure 4). Values of mean and standard deviation of R1, R2 and 1H,15N-NOE were

320

1.4 ± 0.1 s-1, 11.6 ± 5.5 s-1 and -0.29 ± 0.17, respectively. Noteworthy, residues in the hinge region

321

(Leu119, Leu161, Val167 and Gly168) have significantly higher values of R2. In particular, the

322

values for Leu161 and Val167 were 39 ± 2 and 36 ± 6 s-1, respectively, suggesting exchange

323

contributions in the region (Figure 4). As the chemical exchange contributions to R2 relaxation

324

rates are dependent on the chemical shift differences among exchanging states 67, high values of

325

R2 for Leu161 and Val167 are likely caused by the reorientation of the aromatic ring of the nearby

326

Phe183 residue (Figure 3).

327


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

328

329 330

Figure 4.

15N

relaxation measurement. Longitudinal relaxation rates (R1), transverse

331

relaxation rates (R2), 1H,15N-heteronuclear nuclear overhauser enhancement (1H,15N-NOE)

332

were obtained from

333

numbers The values of mean and standard deviation for R1, R2, 1H,15N-NOE were 1.4 ± 0.1

334

s-1, 11.6 ± 5.5 s-1 and -0.29 ± 0.17, respectively.

15N-relaxation

experiments and were plotted against residue


Page 24 of 46

Page 25 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

335


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

336

Mutagenesis studies on yeast ribosomes

337

Structural comparison of B. mori uL10ext with eRF1/ABCE1-bound and eEF2-bound ribosomes

338

revealed significant structural changes in a “hinge” region surrounding Phe183 (Figure 3),

339

resulting the uL10ext domain to undergo a rigid-body movement upon binding of different

340

translation factors (Figure S2). To test the role of this conserved phenylalanine residue, we created

341

a yeast mutant strain expressing a F181A variant of uL10 (Phe181 of yeast uL10 corresponds to

342

Phe183 of B. mori uL10). The strategy of creating the yeast mutant strain is described in Figure

343

5A. In brief, a plasmid expressing a c-Myc-tagged uL10 (pRPP0-myc, Table 3) was transformed

344

to BY4741 to yield the yeast strain KBP01. Western blot showed that both endogenous and c-

345

Myc-tagged uL10 were expressed in KBP01 strain (Figure 5B, lane 2). The endogenous RPP0

346

gene was then knocked-out to create the KBP02 strain (Figure 5B, lane 3). Plasmids expressing

347

wild-type or mutant T7-tagged uL10 (pRPP0-T7, pRPP0-T7-F181A, Table 1) were then

348

transformed to KBP02 to create KBP03-WT and KBP03-F181A strains, respectively. To obtain

349

yeast strains expressing only the T7-tagged uL10, the pRPP0-myc plasmid was removed by FOA

350

counter-selection to create the KBP04-WT and KBP04-F181A strains (Table 2). Western blot

351

analyses confirmed that only the T7-tagged uL10 variants were expressed in the KBP04 strains

352

(Figure 5C). Yeast 80S ribosomes from KBP04-WT and KBP04-F181A strains were purified and

353

assayed for their activities in poly-phenylalanine synthesis (Figure 5D). Interestingly, our results

354

showed that substituting the phenylalanine residue with alanine increased the translation activity

355

by ~33%.

356


Page 26 of 46

Page 27 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

357 358

Figure 5. Translation activity of mutant ribosomes purified from Saccharomyces cerevisiae.

359

(A) Schematic diagram showing the strategy of introducing mutations to uL10. The haploid yeast

360

strain BY4741 was first transformed with the plasmid pRPP0-myc. The genomic copy RPP0 was

361

then knocked out by homologous recombination using a disruption cassette containing a

362

NatRMX4 gene. The plasmid containing mutant uL10 gene was transformed into the uL10-

363

knockout strain. Finally, the plasmid pRPP0-myc was extruded by 5’fluoroorotic acid (5’FOA)

364

screening (B) Western blot analyses confirming the the genomic copy of the RPP0 gene was

365

successfully knocked out in the KBP02 strain. (C) Western blot analyses showing the pRPP0-myc

366

plasmid was removed successfully by FOA counter-selection, leaving the yeast strains expressing

367

only the T7-tagged variants of uL10. (D) Ribosomes carrying a T7-tagged wild-type or mutated

368

uL10 were assayed for poly-phenylalanine synthesis activity as described in Materials and


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

369

Methods. The poly-phenylalanine synthesis activities of mutant ribosomes were plotted as a

370

percentage of that of the wild type ribosome. The mutant F181A caused a ~33% increase of the

371

translation activity when compared to the wild type (p-value=0.0002).

372


Page 28 of 46

Page 29 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

373

DISCUSSION

374

In this study, we combined structural and mutagenesis study to better understand the role of the

375

uL10ext domain in protein translation. During the elongation cycle, eukaryotic ribosome

376

undergoes a number of conformational changes driven by hydrolysis of GTP/ATP

377

functional states

378

structures were determined at resolution < 4 Å (Figure S2)

379

with the binding of GTP-bound elongation factor 1 (eEF1α) and the aminoacyl-tRNA (aa-tRNA)

380

to the ribosomes for the decoding process (Figure S2, PDB: 5LZS

381

cognate tRNA triggers GTP hydrolysis leading to the dissociation of the GDP-bound eEF1α from

382

the ribosomes 73,74. The ribosomes then go through a series of conformational changes to reach a

383

rotated pre-translocation state (PRE)

384

(Figure S2, PDB: 3J7P

385

tRNA/mRNA complex and the ribosomes subsequently reach the post-translocation state (POST)

386

(Figure S2, PDB: 5AJ0

387

ABCE1 are recruited to the ribosomes to eventually form the eRF1/ABCE1-bound state ready for

388

recycle (Figure S2, PDB: 5LZV

389

domain is defined in the eEF2-bound and eRF1/ABCE1-bound ribosomes, but not in the

390

eEF1/aa-tRNA-bound and POST states (Figure S2).

69–72.

68

to yield

Several states were captured by cryo-electron microscopy and their

66) 73,74.

70.

65,66,71,72.

The elongation cycle starts

65) 73,74.

The recognition of

GTP-bound eEF2 then associates with the ribosome

GTP hydrolysis promotes the translocation of the peptidyl-

71) 70,72,73,75,76.

When a stop codon is encountered, release factors and

65) 73,74,77,78.

Among these conformational states, the uL10ext

391 392

Here, we determined the solution structure of the BmuL10ext by NMR spectroscopy and

393

compared it with the structures of uL10 in the eEF2-bound and eRF1/ABCE-bound ribosomes.

394

We showed that the side-chain of the conserved Phe183 residue swings towards the loop between

395

1 and 2 in the eRF1/ABCE1-bound ribosomes, pushing 1/ 2 and 5/ 6 away from the loop


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

396

connecting to the RNA-binding domain (Figure 3A). On the other hand, in BmuL10ext and in

397

eEF2-bound ribosomes, 1/ 2 and 5/ 6 residues pack around Phe183 via hydrophobic

398

interactions with Ala111, Ala115, Pro118, Leu161 and Val167 (Figure 3A).

399 400

The observed structural differences suggest that the uL10ext domain can undergo rigid body

401

movements around the hinge region (Phe183 and the surrounding residues in 1/ 2 and 5/ 6)

402

upon binding of different translation factors (Figure S3A). That side-chain of Phe183 can undergo

403

reorientation was also supported by significant chemical contributions to the R2 relaxation rates

404

for nearby residues Leu161 and Val167 in the BmuL10ext domain. Interestingly, this

405

phenylalanine residue is conserved in eukaryotic but not in archaeal uL10. To test the role of this

406

residue, we created yeast mutant strains expressing a F181A variant of uL10 (Phe181 is the

407

corresponding residue in the yeast sequence). We showed that the F181A substitution in yeast

408

ribosomes increased the translation activity by ~33% (Figure 5). As the conserved phenylalanine

409

residue make a number of hydrophobic interactions with other residues in the hinge region (Figure

410

3), the alanine substitution would reduce the steric hindrance and facilitate the hinge motion of the

411

uL10ext domain, which may play a role in protein translation.

412 413

Conceptually speaking, protein translation requires the binding and release of a series of

414

translation factors to the GTPase-associated centre 2,79. Given that translation factors have different

415

shapes and they bind to the ribosomes at different states, it is conceivable that the ribosomal stalk

416

must change conformations to accommodate different translation factors. We revisited published

417

ribosome structures with resolution better than 4 Å 65,66,71 and found large conformational changes

418

in the base of the ribosomal stalk constituted by uL10, uL11 and the H42-H44 stem loops of 28S


Page 30 of 46

Page 31 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

419

rRNA (Figure S3). During the elongation cycle, the uL10ext domain is structured in the eEF2-

420

bound state but not in the eEF1-bound and the POST states (Figure S2). In the eEF2-bound

421

ribosomes, the uL10ext domain and uL11 make extensive contacts with the bound eEF2 (Figure

422

S3B) 66. In particular, 1 and 3/4 of the uL10ext domain is interacting with the bound eEF2,

423

stabilizing the conformation of the uL10ext domain (Figure S3B). In the eRF1/ABCE1-bound

424

ribosomes, interaction between uL11 and eRF1 causes the H42-H44 stem loops to move towards

425

the eRF1 binding site (Figure S3C and S3D). The conformational changes in the stem loop are

426

propagated to the uL10ext domain via a conserved Arg112 residue. In the eEF2-bound ribosomes,

427

Arg112 form a salt-bridge with the backbone phosphate group of C2014 (Figure S3E). This salt-

428

bridge is broken when the H42-H44 stem loops move away from the uL10ext domain in the

429

eRF1/ABCE1-bound ribosomes. Instead, Arg112 moves towards G1968 forming a salt-bridge

430

with the phosphate group there (Figure S3F) and induces the structural rearrangement around the

431

Phe183 and the hinge motion of the uL10ext domain (Figure 3 & S3A). Our

432

analyses showed that residues in the hinge region, in particular Leu161 and Val167, have

433

significantly faster R2 relaxation rates (Figure 2B) likely due to the reorientation of Phe183 in the

434

uL10ext domain. Taken together with our yeast mutagenesis studies, our results support a model

435

where that the hinge motion of the uL10ext domain is required for recognition of different

436

translation factors during protein translation.

15N

relaxation

437 438

The role of the uL10ext domain in protein translation in eukaryotic ribosomes is also supported

439

by the observations that mutation or deletion of the uL10ext domain reduced the eEF2 dependent

440

GTPase activity and polyphenylalanine synthesis

441

ribosomes 26. It is noteworthy to point out that the uL10ext domain is absent in the bacterial uL10.

25,26,30

and the amount of eEF2 bound to the


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 46

442

Instead, the C-terminal domain of bL12 protein (L12-CTD) in bacterial ribosomes occupies a

443

position similar to that of the uL10ext domain in eukaryotic ribosomes80,81 (Figure S4). bL12-CTD

444

interacts with the elongation factor G and stimulates its GTPase activity 7. Moreover, bacterial

445

uL10 contains a flexible pivot between the spine-helices and the RNA-binding domain 7. uL11

446

that constitutes the base of the ribosome stalk contains a flexible linker between the N-terminal

447

domain and the RNA-binding C-terminal domain82,83. Bacterial bL12 also contains a flexible hinge

448

region84. The flexible regions in these ribosomal proteins facilitate reorientation of their respective

449

domains for binding translation factors

450

dependent GTPase activity on the ribosomal GTPase-associated centre

451

uL10ext domain may play an analogous role in eukaryotic ribosomes in recognition of different

452

translation factors. The uL10ext domain characterized in this study will provide insights for further

453

functional studies of the uL10ext domain.

7,84–86

and play an important role in elongation factors7,85,87.

It is likely that the

454 455

ACCESSION CODES

456

The ensemble of BmuL10ext structures were deposited in Protein Data Bank (accession code:

457

6J3L). Resonance assignments were deposited in BioMagResBank (accession code: 36233). The

458

amino acid sequence of BmuL10 was retrieved from UniprotKB (accession code: Q5UAU1)

459 460 461

ASSOCIATED CONTENTS Supporting Information

462

1H-15N

463

eukaryotic ribosomes; structural changes in the stalk base of eukaryotic ribosomes; structural

correlation map of BmuL10ext; structures of the uL10ext domain in various states of


Page 33 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

464

comparison of the stalk base of eEF2-bound eukaryotic ribosomes and EF-G-bound bacterial

465

ribosomes

466

AUTHOR INFORMATION

467

Corresponding Author

468

*E-mail: [email protected]

469

Funding

470

This work was supported by grants from Research Grants Council (CUHK14122015, AoE/M-

471

403/16, AoE/M-05/12) and Direct Grants from the Research Committee of the Chinese University

472

of Hong Kong.

473 474

ABBREVIATIONS

475

uL10ext, the N-terminal extended protuberant domain of ribosomal protein uL10; eEF1,

476

eukaryotic elongation factor 1; eEF2, eukaryotic elongation factor 2; eRF1, eukaryotic release

477

factor 1; ABCE1, ATP-binding cassette sub-family E member 1; 5’FOA, 5’Fluoroorotic acid;

478

bL12-CTD, the C-terminal domain of bL12; POST, post-translocation state; PRE, pre-

479

translocation state; aa-tRNA, aminoacyl-tRNA; R1, longitudinal relaxation rate; R2, transverse

480

relaxation rate; 1H,15N-NOE, 1H,15N-heteronuclear nuclear overhauser enhancement; GCN2,

481

general

482

dimethylsulphoxide; YPAD, yeast extract adenine dextrose; SD, synthetic dextrose; UTR,

483

untranslated regions; RPP0, DNA sequence of Saccharomyces cerevisiae uL10; ADH1, alcohol

control

nonderepressible

2;

NMR,

nuclear


magnetic

resonance;

DMSO,

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

484

dehydrogenase 1; natRMX4, nourseothricin resistant gene ; TEF1, gene of Saccharomyces

485

cerevisiae eEF1α; RMSD, root-mean-square-displacement from the mean structure.

486


Page 34 of 46

Page 35 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

487

Biochemistry

REFERENCES

488

(1) Wahl, M. C., and Möller, W. (2002) Structure and function of the acidic ribosomal stalk

489

proteins. Curr. Protein Pept. Sci. 3, 93–106.

490

(2) Gonzalo, P., and Reboud, J. P. (2003) The puzzling lateral flexible stalk of the ribosome. Biol.

491

Cell 95, 179–193.

492

(3) Remacha, M., Jimenez-Diaz, A., Santos, C., Briones, E., Zambrano, R., Rodriguez Gabriel, M.

493

A., Guarinos, E., and Ballesta, J. P. (1995) Proteins P1, P2, and P0, components of the eukaryotic

494

ribosome stalk. New structural and functional aspects. Biochem. Cell Biol. 73, 959–968.

495

(4) Lee, K. M., Yusa, K., Chu, L. O., Yu, C. W. H., Oono, M., Miyoshi, T., Ito, K., Shaw, P. C.,

496

Wong, K. B., and Uchiumi, T. (2013) Solution structure of human P1•P2 heterodimer provides

497

insights into the role of eukaryotic stalk in recruiting the ribosome-inactivating protein

498

trichosanthin to the ribosome. Nucleic Acids Res. 41, 8776–8787.

499

(5) Ito, K., Honda, T., Suzuki, T., Miyoshi, T., Murakami, R., Yao, M., and Uchiumi, T. (2014)

500

Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α.

501

Nucleic Acids Res. 42, 14042–14052.

502

(6) Subramanian, A. R. (1975) Copies of proteins L7 and L12 and heterogeneity of the large

503

subunit of Escherichia coli ribosome. J. Mol. Biol. 95, 1–8.

504

(7) Diaconu, M., Kothe, U., Schlünzen, F., Fischer, N., Harms, J. M., Tonevitsky, A. G., Stark, H.,

505

Rodnina, M. V., and Wahl, M. C. (2005) Structural basis for the function of the ribosomal L7/12

506

stalk in factor binding and GTPase activation. Cell 121, 991–1004.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

507

(8) Maki, Y., Hashimoto, T., Zhou, M., Naganuma, T., Ohta, J., Nomura, T., Robinson, C. V., and

508

Uchiumi, T. (2007) Three binding sites for stalk protein dimers are generally present in ribosomes

509

from archaeal organism. J. Biol. Chem. 282, 32827–32833.

510

(9) Uchiumi, T., Traut, R. R., and Wahba, A. J. (1987) Topography and stoichiometry of acidic

511

proteins in large ribosomal subunits from Artemia salina as determined by crosslinking. Proc. Natl.

512

Acad. Sci. 84, 5580–5584.

513

(10) Hagiya, A., Naganuma, T., Maki, Y., Ohta, J., Tohkairin, Y., Shimizu, T., Nomura, T.,

514

Hachimori, A., and Uchiumi, T. (2005) A mode of assembly of P0, P1, and P2 proteins at the

515

GTPase-associated Center in animal ribosome: In vitro analyses with P0 truncation mutants. J.

516

Biol. Chem. 280, 39193–39199.

517

(11) Lee, K. M., Yu, C. W. H., Chan, D. S. B., Chiu, T. Y. H., Zhu, G., Sze, K. H., Shaw, P. C.,

518

and Wong, K. B. (2010) Solution structure of the dimerization domain of ribosomal protein P2

519

provides insights for the structural organization of eukaryotic stalk. Nucleic Acids Res. 38, 5206–

520

5216.

521

(12) Lee, K. M., Yu, C. W. H., Chiu, T. Y. H., Sze, K. H., Shaw, P. C., and Wong, K. B. (2012)

522

Solution structure of the dimerization domain of the eukaryotic stalk P1/P2 complex reveals the

523

structural organization of eukaryotic stalk complex. Nucleic Acids Res. 40, 3172–3182.

524

(13) Krokowski, D., Boguszewska, A., Abramczyk, D., Liljas, A., Tchórzewski, M., and

525

Grankowski, N. (2006) Yeast ribosomal P0 protein has two separate binding sites for P1/P2

526

proteins. Mol. Microbiol. 60, 386–400.

527

(14) Chan, D. S. B., Chu, L. O., Lee, K. M., Too, P. H. M., Ma, K. W., Sze, K. H., Zhu, G., Shaw,


Page 36 of 46

Page 37 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

528

P. C., and Wong, K. B. (2007) Interaction between trichosanthin, a ribosome-inactivating protein,

529

and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses.

530

Nucleic Acids Res. 35, 1660–1672.

531

(15) Too, P. H. M., Ma, M. K. W., Mak, A. N. S., Wong, Y. T., Tung, C. K. C., Zhu, G., Au, S.

532

W. N., Wong, K. B., and Shaw, P. C. (2009) The C-terminal fragment of the ribosomal P protein

533

complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and

534

the ribosome. Nucleic Acids Res. 37, 602–610.

535

(16) Shi, W. W., Tang, Y. S., Sze, S. Y., Zhu, Z. N., Wong, K. B., and Shaw, P. C. (2016) Crystal

536

structure of ribosome-inactivating protein ricin a chain in complex with the C-terminal peptide of

537

the ribosomal stalk protein P2. Toxins 8, 296.

538

(17) Grela, P., Li, X. P., Horbowicz, P., Dźwierzyńska, M., Tchórzewski, M., and Tumer, N. E.

539

(2017) Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain

540

with a prominent role for P1 C-terminus. Sci. Rep. 7: 5608.

541

(18) Murakami, R., Singh, C. R., Morris, J., Tang, L., Harmon, I., Takasu, A., Miyoshi, T., Ito, K.,

542

Asano, K., and Uchiumi, T. (2018) The Interaction between the Ribosomal Stalk Proteins and

543

Translation Initiation Factor 5B Promotes Translation Initiation. Mol. Cell. Biol. 38: e00067-18.

544

(19) Nomura, N., Honda, T., Baba, K., Naganuma, T., Tanzawa, T., Arisaka, F., Noda, M.,

545

Uchiyama, S., Tanaka, I., Yao, M., and Uchiumi, T. (2012) Archaeal ribosomal stalk protein

546

interacts with translation factors in a nucleotide-independent manner via its conserved C terminus.

547

Proc. Natl. Acad. Sci. U. S. A. 109, 3748–3753.

548

(20) Tanzawa, T., Kato, K., Girodat, D., Ose, T., Kumakura, Y., Wieden, H.-J., Uchiumi, T.,


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

549

Tanaka, I., and Yao, M. (2018) The C-terminal helix of ribosomal P stalk recognizes a hydrophobic

550

groove of elongation factor 2 in a novel fashion. Nucleic Acids Res. 46, 3232–3244.

551

(21) Grela, P., Gajda, M. J., Armache, J.-P., Beckmann, R., Krokowski, D., Svergun, D. I.,

552

Grankowski, N., and Tchórzewski, M. (2012) Solution structure of the natively assembled yeast

553

ribosomal stalk determined by small-angle X-ray scattering. Biochem. J. 444, 205–209.

554

(22) Grela, P., Krokowski, D., Gordiyenko, Y., Krowarsch, D., Robinson, C. V., Otlewski, J.,

555

Grankowski, N., and Tchórzewski, M. (2010) Biophysical properties of the eukaryotic ribosomal

556

stalk. Biochemistry 49, 924–933.

557

(23) Wawiórka, L., Molestak, E., Szajwaj, M., Michalec-Wawiórka, B., Mołoń, M., Borkiewicz,

558

L., Grela, P., Boguszewska, A., and Tchórzewski, M. (2017) Multiplication of Ribosomal P-Stalk

559

Proteins Contributes to the Fidelity of Translation. Mol. Cell. Biol. 37: e00060-17.

560

(24) Shimmin, L. C., Ramirez, C., Matheson, A. T., and Dennis, P. P. (1989) Sequence alignment

561

and evolutionary comparison of the L10 equivalent and L12 equivalent ribosomal proteins from

562

archaebacteria, eubacteria, and eucaryotes. J. Mol. Evol. 29, 448–462.

563

(25) Mochizuki, M., Kitamyo, M., Miyoshi, T., Ito, K., and Uchiumi, T. (2012) Analysis of

564

chimeric ribosomal stalk complexes from eukaryotic and bacterial sources: Structural features

565

responsible for specificity of translation factors. Genes to Cells 17, 273–284.

566

(26) Santos, C., and Ballesta, J. P. G. (2005) Characterization of the 26S rRNA-binding domain in

567

Saccharomyces cerevisiae ribosomal stalk phosphoprotein P0. Mol. Microbiol. 58, 217–226.

568

(27) Santos, C., Rodríguez-Gabriel, M. A., Remacha, M., and Ballesta, J. P. G. (2004) Ribosomal

569

P0 protein domain involved in selectivity of antifungal sordarin derivatives. Antimicrob. Agents


Page 38 of 46

Page 39 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

570

Chemother. 48, 2930–2936.

571

(28) Gómez-Lorenzo, M. G., and Garcîa-Bustos, J. F. (1998) Ribosomal P-protein stalk function

572

is targeted by sordarin antifungals. J. Biol. Chem. 273, 25041–25044.

573

(29) Justice, M. C., Ku, T., Hsu, M. J., Carniol, K., Schmatz, D., and Nielsen, J. (1999) Mutations

574

in ribosomal protein L10e confer resistance to the fungal- specific eukaryotic elongation factor 2

575

inhibitor sordarin. J. Biol. Chem. 274, 4869–4875.

576

(30) Naganuma, T., Nomura, N., Yao, M., Mochizuki, M., Uchiumi, T., and Tanaka, I. (2010)

577

Structural basis for translation factor recruitment to the eukaryotic/archaeal ribosomes. J. Biol.

578

Chem. 285, 4747–4756.

579

(31) Inglis, A. J., Masson, G. R., Shao, S., Perisic, O., McLaughlin, S. H., Hegde, R. S., and

580

Williams, R. L. (2019) Activation of GCN2 by the ribosomal P-stalk. Proc. Natl. Acad. Sci. U. S.

581

A. 116, 4946–4954.

582

(32) Kravchenko, O., Mitroshin, I., Nikonov, S., Piendl, W., and Garber, M. (2010) Structure of a

583

two-domain N-terminal fragment of ribosomal protein L10 from Methanococcus jannaschii

584

reveals a specific piece of the archaeal ribosomal stalk. J. Mol. Biol. 399, 214–220.

585

(33) Fiser, A., and Sali, A. (2003) Modeller: generation and refinement of homology-based protein

586

structure models. Methods Enzymol. 374, 461–491.

587

(34) Sikorski, R. S., and Hieter, P. (1989) A System of Shuttle Vectors and Yeast Host Strains

588

Designed for Efficient Manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.

589

(35) Murray, V., Huang, Y., Chen, J., Wang, J., and Li, Q. (2012) A novel bacterial expression

590

method with optimized parameters for very high yield production of triple-labeled proteins.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

591

Methods Mol. Biol. 831, 1–18.

592

(36) Wittekind, M., and Mueller, L. (1993) HNCACB, a High-Sensitivity 3D NMR Experiment to

593

Correlate Amide-Proton and Nitrogen Resonances with the Alpha- and Beta-Carbon Resonances

594

in Proteins. J. Magn. Reson. Ser. B 101, 201–205.

595

(37) Muhandiram, D. R., and Kay, L. E. (1994) Gradient-Enhanced Triple-Resonance Three-

596

Dimensional NMR Experiments with Improved Sensitivity. J. Magn. Reson. Ser. B. 103, 203-216

597

(38) Grzesiekt, S., and Bax, A. (1992) Correlating Backbone Amide and Side Chain Resonances

598

in Larger Proteins by Multiple Relayed Triple Resonance NMR. J. Am. Chem. Soc. 114, 6291–

599

6293.

600

(39) Schleucher, J., Schwendinger, M., Sattler, M., Schmidt, P., Schedletzky, O., Glaser, S. J.,

601

Sørensen, O. W., and Griesinger, C. (1994) A general enhancement scheme in heteronuclear

602

multidimensional NMR employing pulsed field gradients. J. Biomol. NMR 4, 301–306.

603

(40) Marion, D., Driscoll, P. C., Kay, L. E., Wingfield, P. T., Bax, A., Gronenborn, A. M., and

604

Clore, G. M. (1989) Overcoming the overlap problem in the assignment of 1H NMR spectra of

605

larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple

606

quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy:

607

application to. Biochemistry 28, 6150–6156.

608

(41) Grzesiekt, S., Anglister, J., and Bax, A. (1993) Correlation of Backbone Amide and Aliphatic

609

Side-Chain Resonances in 13C/15N-Enriched Proteins by Isotropic Mixing of 13C Magnetization.

610

J. Magn. Reson. Ser. B 101, 114–119.

611

(42) Kay, L. E., Xu, G., Singer, A. U., Muhandiram, D. R., and Forman-Kay, J. D. (1993) A


Page 40 of 46

Page 41 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

612

Gradient-Enhanced HCCH-TOCSY Experiment for Recording Side-Chain

613

Correlations in H2O Samples of Proteins. J. Magn. Reson. Ser. B 101, 333–337.

614

(43) Bax, A., Clore, G. M., and Gronenborn, A. M. (1990) 1H-1H correlation via isotropic mixing

615

of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-

616

enriched proteins. J. Magn. Reson. 88, 425–431.

617

(44) Bodenhausen, G., and Ruben, D. J. (1980) NATURAL ABUNDANCE NITROGEN-15 NMR

618

BY ENHANCED HETERONUCLEAR SPECTROSCOPY. Chem. Phys. Lett. 69, 185–189.

619

(45) Szyperski, T., Neri, D., Leiting, B., Otting, G., and Wüthrich, K. (1992) Support of 1H NMR

620

assignments in proteins by biosynthetically directed fractional

621

323–334.

622

(46) Wider, G., Neri, D., Otting, G., and Wüjthrich, K. (1989) A heteronuclear three-dimensional

623

NMR experiment for measurements of small heteronuclear coupling constants in biological

624

macromolecules. J. Magn. Reson. 85, 426–431.

625

(47) Muhandiram, D. R., Farrow, N. A., Xu, G. Y., Smallcombe, S. H., and Kay, L. E. (1993) A

626

Gradient 13C NOESY-HSQC Experiment for Recording NOESY Spectra of 13C-Labeled Proteins

627

Dissolved in H2O. J. Magn. Reson. Ser. B. 102, 317-321.

628

(48) Zwahlen, C., Gardner, K. H., Sarma, S. P., Horita, D. A., Byrd, R. A., and Kay, L. E. (1998)

629

An NMR experiment for measuring methyl-methyl NOEs in

630

resolution. J. Am. Chem. Soc. 120, 7617–7625.

631

(49) Wider, G., Macura, S., Kumar, A., Ernst, R. R., and Wüthrich, K. (1984) Homonuclear two-

632

dimensional 1H NMR of proteins. Experimental procedures. J. Magn. Reson. 56, 207–234.


13C-labeling.

13C-labeled

1H

and

13C

J. Biomol. NMR 2,

proteins with high

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

633

(50) Johnson, B. A., and Blevins, R. A. (1994) NMR View: A computer program for the

634

visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614.

635

(51) Cornilescu, G., Delaglio, F., and Bax, A. (1999) Protein backbone angle restraints from

636

searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302.

637

(52) Haggis, G. H. (1956) Proton-deuteron exchange in proteins dissolved in heavy water.

638

Biochim. Biophys. Acta 19, 545–546.

639

(53) Linge, J. P., O’Donoghue, S. I., and Nilges, M. (2001) Automated assignment of ambiguous

640

nuclear overhauser effects with ARIA. Methods Enzymol. 339, 71-90.

641

(54) Brünger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grossekunstleve, R. W.,

642

Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and

643

Warren, G. L. (1998) Crystallography & NMR system: A new software suite for macromolecular

644

structure determination. Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905–921.

645

(55) Brunger, A. T. (2007) Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2,

646

2728–2733.

647

(56) Kay, L. E., Torchia, D. A., and Bax, A. (1989) Backbone Dynamics of Proteins As Studied

648

by

649

Nuclease. Biochemistry 28, 8972–8979.

650

(57) Renner, C., Schleicher, M., Moroder, L., and Holak, T. A. (2002) Practical aspects of the 2D

651

15N-{1H}-NOE

652

(58) Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe:

653

A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–

15N

Inverse Detected Heternuclear NMR Spectroscopy: Application to Staphylococcal

experiment. J. Biomol. NMR 23, 23–33.


Page 42 of 46

Page 43 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

654

293.

655

(59) Hill, J., Donald, K. A., Griffiths, D. E., and Donald, G. (1991) DMSO-enhanced whole cell

656

yeast transformation. Nucleic Acids Res. 19, 5791.

657

(60) Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D.

658

(1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of

659

strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132.

660

(61) Manivasakam, P., Weber, S. C., Mcelver, J., and Schiestl, R. H. (1995) Micro-homology

661

mediated PCR targeting in Saccharomyces cerevisiae. Nucleic Acids Res. 23, 2799–2800.

662

(62) Gietz, R. D., Schiestl, R. H., Willems, A. R., and Woods, R. A. (1995) Studies on the

663

transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360.

664

(63) Boeke, J. D., Lacroute, F., and Fink, G. R. (1984) A positive selection for mutants lacking

665

orotidine-5’-phosphate decarboxylase activity in yeast : 5-fluoro-orotic acid resistance. Mol. Gen.

666

Genet. 197, 345–346.

667

(64) Matasova, N. B., Myltseva, S. V., Zenkova, M. A., Graifer, D. M., Vlaclimirov, S. N., and

668

Karpoval, G. G. (1991) Isolation of Ribosomal Subunits Containing Intact rRNA from Human

669

Placenta : Estimation of Functional Activity of 80s Ribosomes. Anal. Biochem. 198, 219–223.

670

(65) Shao, S., Murray, J., Brown, A., Taunton, J., Ramakrishnan, V., and Hegde, R. S. (2016)

671

Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes. Cell 167,

672

1229–1240.

673

(66) Voorhees, R. M., Fernández, I. S., Scheres, S. H. W., and Hegde, R. S. (2014) Structure of

674

the mammalian ribosome-Sec61 complex to 3.4 Å resolution. Cell 157, 1632–1643.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

675

(67) Shapiro, Y. E. (2013) NMR spectroscopy on domain dynamics in biomacromolecules. Prog.

676

Biophys. Mol. Biol. 112, 58–117.

677

(68) Frank, J., and Gonzalez, R. L. (2010) Structure and dynamics of a processive Brownian motor:

678

the translating ribosome. Annu. Rev. Biochem. 79, 381–412.

679

(69) Budkevich, T., Giesebrecht, J., Altman, R. B., Munro, J. B., Mielke, T., Nierhaus, K. H.,

680

Blanchard, S. C., and Spahn, C. M. T. (2011) Structure and dynamics of the mammalian ribosomal

681

pretranslocation complex. Mol. Cell 44, 214–224.

682

(70) Budkevich, T. V, Giesebrecht, J., Behrmann, E., Loerke, J., Ramrath, D. J. F., Mielke, T.,

683

Ismer, J., Hildebrand, P. W., Tung, C.-S., Nierhaus, K. H., Sanbonmatsu, K. Y., and Spahn, C. M.

684

T. (2014) Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific

685

ribosome rearrangement. Cell 158, 121–131.

686

(71) Behrmann, E., Loerke, J., Budkevich, T. V, Yamamoto, K., Schmidt, A., Penczek, P. A., Vos,

687

M. R., Bürger, J., Mielke, T., Scheerer, P., and Spahn, C. M. T. (2015) Structural snapshots of

688

actively translating human ribosomes. Cell 161, 845–857.

689

(72) Flis, J., Holm, M., Rundlet, E. J., Loerke, J., Hilal, T., Dabrowski, M., Bürger, J., Mielke, T.,

690

Blanchard, S. C., Spahn, C. M. T., and Budkevich, T. V. (2018) tRNA Translocation by the

691

Eukaryotic 80S Ribosome and the Impact of GTP Hydrolysis. Cell Rep. 25, 2676-2688.

692

(73) Dever, T. E., and Green, R. (2012) The elongation, termination, and recycling phases of

693

translation in eukaryotes. Cold Spring Harb. Perspect. Biol. 4: a013706.

694

(74) Schuller, A. P., and Green, R. (2018) Roadblocks and resolutions in eukaryotic translation.

695

Nat. Rev. Mol. Cell Biol. 19, 526–541.


Page 44 of 46

Page 45 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

696

(75) Spahn, C. M. T., Gomez-Lorenzo, M. G., Grassucci, R. A., Jørgensen, R., Andersen, G. R.,

697

Beckmann, R., Penczek, P. A., Ballesta, J. P. G., and Frank, J. (2004) Domain movements of

698

elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J.

699

23, 1008–1019.

700

(76) Taylor, D. J., Nilsson, J., Merrill, A. R., Andersen, G. R., Nissen, P., and Frank, J. (2007)

701

Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in

702

translocation. EMBO J. 26, 2421–2431.

703

(77) Jackson, R. J., Hellen, C. U. T., and Pestova, T. V. (2012) Termination and post-termination

704

events in eukaryotic translation. Adv. Protein Chem. Struct. Biol. 86, 45–93.

705

(78) Buskirk, A. R., and Green, R. (2017) Ribosome pausing, arrest and rescue in bacteria and

706

eukaryotes. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 372: 20160183

707

(79) Liljas, A., and Sanyal, S. (2018) The enigmatic ribosomal stalk. Q. Rev. Biophys. 51: e12.

708

(80) Harms, J. M., Wilson, D. N., Schluenzen, F., Connell, S. R., Stachelhaus, T., Zaborowska, Z.,

709

Spahn, C. M. T., and Fucini, P. (2008) Translational Regulation via L11: Molecular Switches on

710

the Ribosome Turned On and Off by Thiostrepton and Micrococcin. Mol. Cell 30, 26–38.

711

(81) Gao, Y., Selmer, M., Dunham, C. M., Weixlbaumer, A., and Kelley, A. C. (2009) The

712

structure of the ribosome with elongation factor G trapped in the post-translocational state. Science

713

326, 694–699.

714

(82) Conn, G. L., Draper, D. E., Lattman, E. E., and Gittis, A. G. (1999) Crystal structure of a

715

conserved ribosomal protein-RNA complex. Science 284, 1171–1174.

716

(83) Wimberly, B. T., Guymon, R., McCutcheon, J. P., White, S. W., and Ramakrishnan, V. (1999)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

717

A Detailed View of a Ribosomal Active Site. Cell 97, 491–502.

718

(84) Mulder, F. A. A., Bouakaz, L., Lundell, A., Venkataramana, M., Liljas, A., Akke, M., and

719

Sanyal, S. (2004) Conformation and dynamics of ribosomal stalk protein L12 in solution and on

720

the ribosome. Biochemistry 43, 5930–5936.

721

(85) Agrawal, R. K., Linde, J., Sengupta, J., Nierhaus, K. H., and Frank, J. (2001) Localization of

722

L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation.

723

J. Mol. Biol. 311, 777–787.

724

(86) Helgstrand, M., Mandava, C. S., Mulder, F. A. A., Liljas, A., Sanyal, S., and Akke, M. (2007)

725

The Ribosomal Stalk Binds to Translation Factors IF2, EF-Tu, EF-G and RF3 via a Conserved

726

Region of the L12 C-terminal Domain. J. Mol. Biol. 365, 468–479.

727

(87) Dey, D., Oleinikov, A. V., and Traut, R. R. (1995) The hinge region of Escherichia coli

728

ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis. Biochimie 77, 925–

729

930.

730 731

For Table of Contents only

732 733


Page 46 of 46

Structural and mutagenesis studies evince the role of the extended

Recommend Documents