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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

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Biochemistry

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Structural and mutagenesis studies evince the role of the

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extended protuberant domain of ribosomal protein uL10 in

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protein translation

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

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

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† School

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

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

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Kong, China

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§ Division

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

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Bay, Hong Kong, China ‡ Department

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

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Biochemistry

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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centre during protein synthesis.

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INTRODUCTION

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

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

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

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

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

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

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

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

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2 to 3 copies of bL12 homodimers

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

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in eukaryotic ribosomes

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

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

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

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

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

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

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

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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

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

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8,24,25

(Figure 1).

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Biochemistry

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

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

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

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

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

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

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

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

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

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initiation factor 2α in response to stress 31

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Crystal structure of an archaeal uL10 from Methanococcus jannaschii was determined

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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biological role of the uL10ext domain was discussed.

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Biochemistry

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

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

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

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

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

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

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motif SDxDMGFxLFx responsible for binding translation factors and ribosome inactivating

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

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

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

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

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

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

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

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3JSY 32 and 3A1Y 30 as templates.

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Biochemistry

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MATERIALS AND METHODS

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Plasmid construction

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For Escherichia coli expression

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

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

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

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

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thrombin cutting site.

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For Saccharomyces cerevisiae expression

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

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

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

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

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

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

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

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

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

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

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plasmids used are listed in Table 1.

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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

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NMR sample preparations

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

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

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

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

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

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

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Biochemistry

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

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

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

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

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

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

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

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

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

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

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

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

148 149

Structure determination

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

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

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HNCACB

153

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

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

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13C-labeled

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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-

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

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

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from hydrogen/deuterium-exchange experiments

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

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

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

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

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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

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15N

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

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

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

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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,

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

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

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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-

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

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

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

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

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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

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

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

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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.

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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

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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

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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

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Biochemistry

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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

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

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

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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

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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

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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

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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.

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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

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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

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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

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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.

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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

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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

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Biochemistry

335

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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

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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

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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

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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

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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

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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

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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

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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

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magnetic

resonance;

DMSO,

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484

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

485

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

486

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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

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930.

730 731

For Table of Contents only

732 733

ACS Paragon Plus Environment

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