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Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

ABSTRACT: Unexpected experimental outcomes led us to hypothesize that synonymously recoding segments of the M. tuberculosis (Mtb) rpoB gene (rv0667) caused the translation of many smaller protein isoforms from translation initiations sites within the recoded open reading frame (ORF). To test this hypothesis, we labeled the N-termini of Mtb rpoB expressed heterologously in the closely related model system M. smegmatis and identified peptides of rpoB carrying the N-terminal label by mass spectrometry. We found an increase in the detection of N-terminally labeled peptides that mapped to recoded regions of the rpoB gene variants, and thus inferred that recoding was linked to the generation of translation initiation sites within the ORF. We also observed a smaller number of N-terminally labeled peptides mapping to the middle of the wild-type ORF, which we interpreted to indicate that some intragenic translation initiation could possibly occur in the native gene or, more likely, arise from heterologous gene expression.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Mycobacterium Smegmatis Str. Mc2 155 (ncbitaxon:246196) Mycobacterium Tuberculosis (ncbitaxon:1773)

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000089557 | MassIVE | Fri May 27 11:26:00 BST 2022

REPOSITORIES: MassIVE

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Project description:Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

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Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

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