Accessibility of the Shine–Dalgarno sequence dictates N-terminal codon bias in<i>E. coli</i>

Bhattacharyya, Sanchari; Jacobs, William M.; Adkar, Bharat V.; Yan, Jin; Zhang, Wenli; Shakhnovich, Eugene I.

doi:10.1101/195727

Cited by 9 publications

(12 citation statements)

References 49 publications

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“…This interpretation rests upon the assumption that most mRNAs are stable enough to contribute to measurements of steady-state abundance. There are various reports of mutations or modifications that affect mRNA levels and protein levels differently (18,19,54,66,69), supporting the idea that translation efficiency can indeed be estimated by comparing protein levels to mRNA levels. Using this assumption and definition of translation efficiency, the Mtb data imply that there is no global difference in translation efficiency for leadered vs. leaderless transcripts.…”

Section: Downloaded Frommentioning

confidence: 88%

The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis

et al. 2020

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Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5′ untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis, the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5′ UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5′ UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5′ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5′ UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium’s survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5′ untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5′ untranslated regions and are unusually prevalent in mycobacteria.

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Section: Downloaded Frommentioning

confidence: 88%

The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis

et al. 2020

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“…Inactivation of mRNA by intramolecular secondary structures is a well-acknowledged cause for inefficient translation in cell-free systems (58,79,90). Intramolecular base pairing occluding the Shine-Dalgarno (SD) sequence can greatly reduce the probability of translation initiation and affect protein levels in vivo (91). Whereas tight coupling between the rate of transcription and translation initiation limits formation of inhibitory RNA structures and ensures efficient usage of mRNA, the level of cooperation between these two processes is low in the T7 RNA polymerasebased PURE system.…”

Section: Discussionmentioning

confidence: 99%

Modelling cell-free RNA and protein synthesis with minimal systems

et al. 2019

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DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (Protein synthesis Using Recombinant Elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, it becomes urgent to improve the system's performance, and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNAhydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.

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“…The prototypical example is represented by riboswitches in which binding to a metabolite results in structural changes within the 5′ UTR that inhibit gene expression (50). Bacteria have evolved to minimize binding between the CDS and respective SD sequences to promote efficient translation initiation and thus enhance fitness (51). Nevertheless, although much less common, long-distance cis-interactions between the CDS and SD sequences have been reported to influence expression of genes important for thermostresses or growth rate (52)(53)(54).…”

Section: Discussionmentioning

confidence: 99%

The sRNA DicF integrates oxygen sensing to enhance enterohemorrhagicEscherichia colivirulence via distinctive RNA control mechanisms

Melson

Kendall

2019

Proc. Natl. Acad. Sci. U.S.A.

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To establish infection, enteric pathogens integrate environmental cues to navigate the gastrointestinal tract (GIT) and precisely control expression of virulence determinants. During passage through the GIT, pathogens encounter relatively high levels of oxygen in the small intestine before transit to the oxygen-limited environment of the colon. However, how bacterial pathogens sense oxygen availability and coordinate expression of virulence traits is not resolved. Here, we demonstrate that enterohemorrhagicEscherichia coliO157:H7 (EHEC) regulates virulence via the oxygen-responsive small RNA DicF. Under oxygen-limited conditions, DicF enhances global expression of the EHEC type three secretion system, which is a key virulence factor required for host colonization, through the transcriptional activator PchA. Mechanistically, thepchAcoding sequence (CDS) base pairs with the 5′ untranslated region of the mRNA to sequester the ribosome binding site (RBS) and inhibit translation. DicF disruptspchA cis-interactions by binding to thepchACDS, thereby unmasking thepchARBS and promoting PchA expression. These findings uncover a feed-forward regulatory pathway that involves distinctive mechanisms of RNA-based regulation and that provides spatiotemporal control of EHEC virulence.

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Accessibility of the Shine–Dalgarno sequence dictates N-terminal codon bias inE. coli

Cited by 9 publications

References 49 publications

The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis

The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis

Modelling cell-free RNA and protein synthesis with minimal systems

The sRNA DicF integrates oxygen sensing to enhance enterohemorrhagicEscherichia colivirulence via distinctive RNA control mechanisms

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