Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Beck, Heather J.; Janssen, Gary R.

doi:10.1128/jb.00091-17

Cited by 10 publications

(7 citation statements)

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“…We first compiled a database of 9862 characterized genetic systems, collected from 15 publications, ,,,,− whereby 7 unique heterologous reporter proteins (mRFP1, super folder GFP, CFP, YFP, LacZ, Luciferase, and NanoLuc) were expressed using engineered promoters and ribosome binding sites in 6 different bacterial hosts ( E. coli , B. subtilis 168, C. glutamicum , S. typhimurium LT2, B. thetaiotaomicron , and P. fluorescens ). The genetic systems’ reporter expression levels were individually characterized using flow cytometry or spectrophotometry, or using FlowSeq, a technique that uses binned cell sorting, bar-coding, and next-generation sequencing to measure the protein levels, and thus the transcription and translation rates of a library of genetic systems .…”

Section: Resultsmentioning

confidence: 99%

An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models

2020

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Gene expression models greatly accelerate the engineering of synthetic metabolic pathways and genetic circuits by predicting sequence-function relationships and reducing trial-and-error experimentation. However, developing models with more accurate predictions is a significant challenge, even though they are essential to engineering complex genetic systems. Here we present a model test system that combines advanced statistics, machine learning, and a database of 9862 characterized genetic systems to automatically quantify model accuracies, accept or reject mechanistic hypotheses, and identify areas for model improvement. We also introduce Model Capacity, a new information theoretic metric that enables correct model comparisons across datasets. We demonstrate the model test system by comparing six models of translation initiation rate, evaluating 100 mechanistic hypotheses, and uncovering new sequence determinants that control protein expression levels. We applied these results to develop a biophysical model of translation initiation rate with significant improvements in accuracy. Automated model test systems will dramatically accelerate the development of gene expression models, and thereby transition synthetic biology into a mature engineering discipline..

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Section: Resultsmentioning

confidence: 99%

An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models

2020

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“…Since strengthening the ptrB SD sequence alleviated the requirement for the 5=-uAUG, it is apparent that recruitment of a 70S ribosome by the 5=-uAUG compensates for a poor ptrB SD sequence. Strikingly, replacing the 5=-UTR of other mRNAs with that from ptrB conferred a similar dependence on the ptrB 5=-uAUG, indicating that the features within the ptrB 5=-UTR are sufficient to control downstream gene expression (16).…”

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confidence: 88%

“…While a variety of noncanonical translation initiation mechanisms have been described, Beck and Janssen have recently described yet another variation of noncanonical translation initiation in E. coli (16). Previous work from this group identified several examples of uORFs positioned at the 5= terminus that influenced expression of the corresponding downstream cistron (17).…”

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confidence: 99%

“…Although increased expression depended on the 5=-terminal AUG codon (5=-uAUG), the uORF was not efficiently translated (17). In their most recent study, Beck and Janssen explored this fascinating translation initiation mechanism (16). Notably, instead of relying on translation of the 5=-uORF, the 5=-uAUG functioned as a 70S ribosome recognition signal to attract ribosomes to ptrB mRNA, which facilitated efficient translation of the ptrB coding sequence further downstream.…”

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confidence: 99%

“…A model was proposed to explain the mechanism of ptrB regulation (16). In this model, the 5=-uAUG acts as a recognition signal and binding platform to attract 70S ribosomes to the transcript, thereby increasing the localized concentration of ribosomes on the ptrB transcript.…”

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Noncanonical Translation Initiation Comes of Age

Babitzke

O’Connor

2017

J Bacteriol

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The canonical translation initiation mechanism involves base pairing between the mRNA and 16S rRNA. However, a variety of identified mechanisms deviate from this conventional route. Beck and Janssen (J Bacteriol 199:e00091-17, 2017, https://doi.org/10.1128/JB.00091-17) have recently described another noncanonical mode of translation initiation. Here, we describe how this process differs from previously reported mechanisms, with the hope that it will foster increased awareness of the diversity of regulatory mechanisms that await discovery. The standard model for initiation of translation in bacteria involves base pairing between a Shine-Dalgarno (SD) sequence upstream of the start codon and the complementary anti-SD (aSD) sequence at the 3= end of 16S rRNA in a 30S ribosomal subunit. This mode of initiation, where the SD is a major component of the ribosome binding site (RBS), is widespread in bacteria and archaea and has until recently been inferred to be the dominant initiation pathway. The model posits that the SD-aSD interaction increases the local concentration of 30S ribosomal subunits in the vicinity of the initiation codon, facilitating subsequent events in initiation complex formation (1). Systematic approaches in Escherichia coli have demonstrated the influence of SD length and its distance from the initiation codon (2). In general, longer SD elements support increased expression; however, the relationship is nonlinear, and binding affinity alone does not fully explain the observed protein expression levels. Weaker SD sequences are particularly susceptible to the effects of mRNA decay and transcription termination (3), while extended (8-to 10-nucleotide [nt]) SD sequences inhibit translation, presumably since such sequences trap the ribosome on the RBS (4). Among E. coli genes, the average SD length is 6.3 nt (5), and the average spacing between the SD and initiation codon is 4.4 nt (4). The SD sequences from Bacillus subtilis, in general, are longer than their E. coli counterparts (6), a property that may be linked to the absence of a large ribosomal protein bS1 in many Gram-positive organisms (7).30S subunit binding and effective initiation is heavily influenced by the mRNA structure surrounding the RBS. Many of the gene regulatory mechanisms that target initiation operate by altering the local RNA structure encompassing SD sequences. These mechanisms include translational coupling, where translation of an upstream open reading frame (uORF) disrupts the RNA structure surrounding the downstream RBS. The binding of small regulatory RNAs or RNA binding proteins, metabolites (in riboswitches), or increases in temperature (in thermosensors) can all influence the local RNA structure, with corresponding effects on initiation. An additional model is required to explain how certain mRNAs with RBSs sequestered in stable structures can be translated effectively. The "standby binding" model posits that 30S subunits bind first to single-stranded RNA flanking the structured, RBS-containing element. The bou...

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Translation Control byRNA

Goldman¹

2019

Encyclopedia of Life Sciences

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Translation control by ribonucleic acid (RNA) refers to the process by which protein synthesis is regulated by structural elements in RNA (primary, secondary or tertiary), often cis ‐acting in the messenger RNA encoding the protein being synthesised, and also trans ‐acting in a number of systems. This article covers the historical early recognition of translation control by RNA in the RNA bacteriophage and extends the concept to include many additional examples of secondary structure control, or ‘riboswitches’, and other methods of controlling access by ribosomes to messenger ribonucleic acid (mRNA), including the role of small regulatory RNAs. Effects of RNA primary sequences on translation are also covered, including initiation signals, recoding (e.g. translational frameshifts), codon bias, translational attenuation and antisense regulation. mRNA stability is also considered, as well as an RNA‐based mechanism to facilitate translation termination, transfer‐messenger ribonucleic acid (tmRNA). Key Concepts mRNA secondary/tertiary structure can obscure or expose translation start sites and can be altered by conditions in the cell to regulate translation initiation (riboswitches). Translational coupling requires translation of an upstream gene before ribosomes can translate the downstream gene. Often used to promote equimolar levels of proteins that are utilised in comparable amounts (enzyme subunits), or for inhibition of more than one gene in an operon (ribosomal protein operons). Small RNAs, generally antisense RNA, can bind to mRNA to inhibit translation starts, or in some cases, to facilitate translation starts. Often requires a protein chaperone‐like Hfq. Antisense RNAs often lead to degradation of the RNA target. In eukaryotes, this is termed ‘ribonucleic acid interference’ (RNAi). Primary sequences in mRNA can also affect translation efficiency, including choice of start codon, or quality of the ribosome‐binding site, or as a target for a repressor protein (translational ‘operators’). Primary sequences in mRNA can in some instances trigger ‘recoding’, in which the mRNA is translated in an unexpected way, such as causing translational frameshifts, or incorporation of selenocysteine in response to a UGA codon. In the phenomenon of ‘translational attenuation’, the primary sequence encodes an inhibitory peptide that potentiates the ribosome to be inhibited by otherwise sub‐lethal concentrations of antibiotic, thereby exposing a downstream translation start for the antibiotic resistance gene. Codon bias can affect the efficiency of translation, where common codons in a given organism are almost exclusively used for highly expressed proteins, and rare codons for that organism used at a much higher frequency in poorly expressed proteins. Usually correlates to cognate tRNA levels. mRNA stability is a determinant of the amount of protein expressed; stability can be affected by both cis ‐ and trans ‐acting elements. tmRNA (transfer‐messenger ribonucleic acid) is a mechanism in bacteria for orderly termination of translation when there is a vacant A site on the ribosome.

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Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Cited by 10 publications

References 55 publications

An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models

An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models

Noncanonical Translation Initiation Comes of Age

Translation Control byRNA

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