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Application of sorting and next generation sequencing to study 5?-UTR influence on translation efficiency in Escherichia coli.

ABSTRACT: Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5? untranslated regions (5?-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5?-UTR sequences and introduced random and designed mutations into natural and artificially selected 5?-UTRs. Several distinct properties could be ascribed to a group of 5?-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5?-UTRs.

SUBMITTER: Evfratov SA

PROVIDER: S-EPMC5389652 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli.

Evfratov Sergey A SA Osterman Ilya A IA Komarova Ekaterina S ES Pogorelskaya Alexandra M AM Rubtsova Maria P MP Zatsepin Timofei S TS Semashko Tatiana A TA Kostryukova Elena S ES Mironov Andrey A AA Burnaev Evgeny E Krymova Ekaterina E Gelfand Mikhail S MS Govorun Vadim M VM Bogdanov Alexey A AA Sergiev Petr V PV Dontsova Olga A OA

Nucleic acids research 20170401 6

Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was tran ...[more]

PMID: 27899632

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Project description:Translational regulation was investigated at the genome-scale in Escherichia coli cells. Using the polysome profiling method, the ribosome occupancy (RO) and ribosome density (RD) of different mRNA copies were determined for several hundred mRNAs during the exponential- and stationary-phases, providing the most complete characterization of such regulation in E. coli. Although for most genes, nearly all mRNAs (>90%) were undergoing translation, they were loaded with far fewer than the theoretical maximum number of ribosomes, suggesting translation limitation at the initiation step. Multiple linear regression was used to identify key intrinsic factors involved in the genome-wide regulation of RO and RD (i.e., open reading frame GC%, protein function, and localization). Unexpectedly, mRNA concentration, a factor that depends on cell physiology, was predicted to positively regulate RO and RD during the exponential- and stationary-phases. Using a set of selected genes controlled by an inducible promoter, we confirmed that increasing the mRNA concentration upon transcription induction led to increases in both RO and ribosome load. The fact that this relationship between mRNA concentration and translation parameters was also effective when E. coli cells naturally adapted to carbon source changes demonstrates its physiological relevance. This work demonstrated that translation regulation is positively controlled by transcript availability. This new mechanism contributed to the codirectional regulation of transcription and translation with synergistic effects on gene expression and provided a systemic understanding of E. coli cell function. IMPORTANCE The process of gene expression is divided into translation and transcription. Considerable efforts have been made in bacteria to characterize the mechanisms underlying translational regulation and identify the regulatory factors for particular mRNAs. However, to understand bacterial physiology and adaptation, it is important to elucidate genome-wide translational regulation and examine its coordination with transcriptional regulation. Here, we provided a genome-wide picture of translational regulation in Escherichia coli. For most genes, nearly all mRNA copies were found to undergo translation but were loaded with a low number of ribosomes. We showed that mRNA concentration had a positive effect on translation regulation, linking translational regulation to transcriptional regulation as well as to cell physiology and growth conditions. The codirectional regulation of transcription and translation had synergistic effects on gene expression, contributing to E. coli cell function optimization. This finding could be used in biotechnology to optimize strategies for recombinant protein synthesis.

Project description:To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm5s2U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation.IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm5s2U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm5s2U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis.

Dataset Information

Application of sorting and next generation sequencing to study 5?-UTR influence on translation efficiency in Escherichia coli.

Publications

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli.

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