Transcriptional Interference in Convergent ... - ACS Publications

Jun 26, 2016 - the transcriptional interference mechanisms of antisense roadblock and. RNA polymerase traffic in a set of convergent promoters as nove...
0 downloads 0 Views 2MB Size
Subscriber access provided by - Access paid by the | UCSB Libraries

Letter

Transcriptional Interference in convergent promoters as a means for Tunable Gene Expression Antoni Escalas Bordoy, Usha S. Varanasi, Colleen M Courtney, and Anushree Chatterjee ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00223 • Publication Date (Web): 26 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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 free 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 accessible to all readers and 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.

ACS Synthetic Biology 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 30

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

ACS Synthetic Biology

1

Transcriptional Interference in convergent promoters as a means for Tunable Gene Expression

2

Antoni E. Bordoy1, Usha S. Varanasi1, Colleen M. Courtney1 and Anushree Chatterjee1,2,*

3

1

4

Boulder, 3415 Colorado Avenue, UCB 596, CO 80303, USA.

Department of Chemical and Biological Engineering, 2BioFrontiers institute, University of Colorado,

5 6

ABSTRACT: An important goal of synthetic biology involves the extension and standardization of

7

novel biological elements for applications in medicine and biotechnology. Transcriptional

8

interference, occurring in sets of convergent promoters, offers a promising mechanism for building

9

elements for the design of tunable gene regulation. Here, we investigate the transcriptional

10

interference mechanisms of antisense roadblock and RNA polymerase traffic in a set of convergent

11

promoters as novel modules for synthetic biology. We show examples of elements, including

12

antisense roadblock, relative promoter strengths, inter-promoter distance, and sequence content

13

that can be tuned to give rise to repressive as well as cooperative behaviors, therefore resulting in

14

distinct gene expression patterns. Our approach will be useful towards engineering new biological

15

devices and will bring new insights to naturally occurring cis-antisense systems. Therefore, we are

16

reporting a new biological tool that can be used for synthetic biology.

17

KEY WORDS: Antisense transcription, transcriptional interference, gene regulation, antisense

18

roadblock, genetic switches, RNA polymerase collision.

19 20

Rational design of new biological systems that allow for better gene regulation has been of

21

outstanding interest in synthetic biology. Their various purposes are to improve biosensing, biofuel and

22

pharmaceutical production, and develop gene therapies that target superbugs and diseases like cancer1.

23

The first synthetic genetic devices designed to improve the control of gene expression, the toggle switch2

24

and repressilator3, paved the way for more complex transcriptional networks interconnected by different

1 ACS Paragon Plus Environment

ACS Synthetic Biology

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

25

repressor/activator-promoter architectures that enabled construction of genetic switches4,5, oscillators6,

26

memory elements7, logic gates7,8, RNA devices9,10 and quorum sensing systems11,12. To achieve such

27

complex cellular responses a vast range of orthogonal regulatory parts have been rationally designed

28

including promoters13, proteins14 and riboregulators15. Despite such great advancements, most of synthetic

29

biology devices that implement regulation via proteins, RNA or both16, consist of separated,

30

interconnected transcriptional units with trans-acting elements, thus requiring coordination between

31

multiple biological parts often leading to high metabolic costs17. Here, inspired by naturally occurring

32

systems, we report a new category of synthetic biological devices that are connected and regulate gene

33

expression through the process of transcriptional interference during cis-antisense transcription. Since this

34

form of regulation occurs during the process of transcription, it reduces the time scale of the regulatory

35

response upon a certain stimulus and lowers the associated cellular energetic cost.

36

Cis-antisense transcription arises when a pair of promoters is arranged in convergent orientation

37

(one in the sense orientation and one in the antisense orientation) via a mechanism termed transcriptional

38

interference18 (TI) that has been observed to play key regulatory role in both prokaryotes19–22 and

39

eukaryotes23–26. Presence of TI (reviewed in ref. 28) at various promoter arrangements has been shown to

40

be ubiquitous20,27–29 and it is therefore believed to be conserved over evolution30. Additionally,

41

cis-antisense transcription allows for significant DNA compression, i.e. it reduces the genomic footprint31.

42

Previously we have shown that cis-antisense transcription has a built-in regulatory role in conferring

43

biological systems with robust bistable genetic switch-like characteristics19,22,32,33. Despite the widespread

44

occurrence of such promoter arrangements, little effort has gone into taking advantage of its regulatory

45

mechanisms for designing synthetic biology devices. Only recently, the CRISPR-dCas9 system has been

46

used to create genetic switches using the TI mechanism of roadblock34. Here, to the best of our

47

knowledge, we present the first attempt to use synthetic TI constructs to understand how the different

48

mechanisms of TI can be integrated to achieve regulation of gene expression. In order to do so, we take

49

advantage of the TI mechanisms of (i) Roadblock, where a DNA bound protein blocks movement of an

2 ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

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

ACS Synthetic Biology

50

elongating RNA polymerase (RNAP)35 (Fig. 1a) and (ii) RNAP traffic, that can either increase the local

51

RNAP concentration at the convergent promoters, or cause RNAP collision in which two opposing

52

elongation complexes (ECs) collide while traversing the overlapping DNA between two convergent

53

promoters often producing truncated transcripts19 (Fig. 1b). Collision between two opposing ECs is

54

influenced by build-up of torsional stress during movement of a transcribing EC on DNA, which is

55

preceded by positive super-coiling and followed by negative super-coiling36, resulting in either RNAP

56

stalling or RNAP backtracking37,38. Although it has been shown that transcription bubbles can pass by one

57

another39, there is sufficient evidence that approach of two convergent ECs causes RNAP to fall off the

58

DNA with consequent production of truncated RNAs19. Since both roadblock and collision affect RNAP

59

traffic over the shared DNA between the convergent promoters, unraveling the nature of these forms of

60

regulation will enable us to use these new transcription-based regulated devices as powerful tools for both

61

scientific research and biotechnology. We show that experimental parameters including antisense

62

roadblock, relative promoter strengths, inter-promoter distance, and RNAP residence time in the

63

overlapping DNA can be used to build distinct expression patterns both in sense and antisense genes.

64



65

Inspired by naturally occurring systems, we arranged two biological components: (i) inducible

66

synthetic PLTet and PLLac promoters regulated by repressor proteins TetR and LacI40, respectively, and

67

(ii) overlapping DNA of various lengths containing naturally occurring bacterial sequences41–45,

68

separating PLTet and PLLac, in order to construct sets of convergent promoters to investigate effect of RNAP

69

traffic and roadblock (Fig. 2a, Supporting Text 1, Table S1-S2). In our experiments, PLTet and PLLac drive

70

expression of green fluorescent protein (GFP) and red fluorescent protein (mCherry), respectively, which

71

provide a visible read out of sense-antisense gene expression. Although the use sense and antisense terms

72

is relative to both strands of DNA, hereafter we refer to sense and antisense strand as the ones that contain

73

PLtet and PLLac promoters, respectively. Various plasmid constructs were harbored by four different strains

74

of model bacterial organism E. coli that expressed either both TetR and LacI repressors (DH5αZ1), only

RESULTS AND DISCUSSION

3 ACS Paragon Plus Environment

ACS Synthetic Biology

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

75

TetR (DH5α-TetR), only LacI (C600Zi), or none of the repressors (DH5α-WT), in order to study extreme

76

cases of promoter activity depending on the repressors expressed (Fig. 2b, Fig. S1). The transcriptional

77

rates of the convergent, orthogonal PLTet and PLLac promoters were changed by inducing them with

78

different levels of anhydrous tetracycline (aTc) and Isopropyl β-D-1-thiogalactopyranoside (IPTG),

79

respectively. Single sense and antisense promoter constructs driving expression of respective reporter

80

genes were designed to serve as controls (Fig. 2c, d, Supporting Text 1). Upon addition of only one

81

inducer, one promoter becomes active while the other remains inactive. We hypothesize activity of the

82

induced promoter can be regulated by the TI mechanism of antisense roadblock whereby the neighboring

83

repressor protein, which is bound to the DNA, impedes the movement of the transcribing RNAP that, in

84

some cases, leads to failed transcription35. We further hypothesize that the sequence content of the

85

overlapping DNA between the convergent promoters can be used as a tunable synthetic biology module,

86

to control gene expression to build various types of genetic switches. Thus, here we present various

87

examples of systems where tunability of gene expression can be achieved due to transcriptional processes.

88

During convergent transcription both sense and antisense transcripts share complementary regions

89

that can potentially participate in RNA interactions. To confirm that the mechanisms being investigated

90

are related to TI and to rule out role antisense RNA interactions, we measured protein expression in a

91

dual-plasmid system containing sense construct, pAE_T145 expressing sense RNA fused to GFP

92

transcript, and the corresponding antisense construct with the complementary overlapping DNA of the

93

same length, pAE_L145_R expressing antisense RNA fused to mCherry transcript in trans (Supporting

94

Text 1, Fig. S1), in strain C600Zi (Supporting Text 2) at various IPTG concentrations. We observed

95

significant changes in mCherry expression at both 0.05 and 0.5 mM IPTG (p-value