Polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression in mycobacteria

Abstract

24 25 Genome-wide transcriptomic analyses have revealed abundant expressed short open 26 reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins 27 they encode, are functional remains an open question. One quarter of mycobacterial 28 mRNAs are leaderless, meaning the RNAs begin with a 5'-AUG or GUG initiation 29 codon. Leaderless mRNAs often encode an unannotated short ORF as the first gene 30 of a polycistronic transcript. Consecutive cysteine codons are highly 31 overrepresented in mycobacterial leaderless short ORFs. Here we show that 32 polycysteine-encoding leaderless short ORFs function as cysteine-responsive 33 attenuators of operonic gene expression. Through detailed mutational analysis, we 34 show that one such polycysteine-encoding short ORF controls expression of the 35 downstream genes by causing ribosome stalling under conditions of low cysteine. 36 Ribosome stalling in turn blocks mRNA secondary structures that otherwise 37 sequester the Shine-Dalgarno ribosome-binding site of the 3'gene. This translational 38 attenuation does not require competing transcriptional terminator formation, a 39 mechanism that underlies traditional amino acid attenuation systems. We further 40 assessed cysteine attenuation in Mycobacterium smegmatis using mass spectrometry 41 to evaluate endogenous proteomic responses. Notably, six cysteine metabolic loci 42 that have unannotated polycisteine-encoding leaderless short ORF architectures 43 responded to cysteine supplementation/limitation, indicating that cysteine-44 responsive attenuation is widespread in mycobacteria. Individual leaderless short 45 ORFs confer independent operon-level control, while their shared dependence on 46 cysteine ensures a collective response. Bottom-up regulon coordination is the 47 antithesis of traditional top-down master regulator regulons and illustrates one 48 utility of the many unnanotated short ORFs expressed in bacterial genomes. 49 50 129We next tested whether cysteine-dependent regulation of Ms5788 requires translation of 130 the upstream Ms5788A LL-sORF. We mutated the GUG translation initiation codon of 131Ms5788A to ACC in the context of the luciferase reporter, to prevent ribosome loading. 132This non-start mutation reduced luciferase expression, and abolished attenuation ( Fig 1B 133 compare i vs ii). These data indicate that translation of Ms5788A is required for cysteine-134 dependent regulation of Ms5788, and that in the absence of Ms5788A translation, 135 expression of Ms5788 is locked in an attenuated state. We speculated that ribosome 136

Mycobacterium tuberculosis VapC4 toxin engages small ORFs to initiate an integrated oxidative and copper stress response

“…We expected up-regulation of cysA2 and sseC2 in cells expressing VapC4, consistent with the luciferase reporter experiments for M. smegmatis Ms5788A (15). Indeed, RNA-seq performed on RNA harvested 24 and 72 h after VapC4 induction showed significant (all P values < 0.0006) increases in both cysA2 and sseC2 transcripts relative to the uninduced controls (Fig.…”

“…widespread ribosome stalling at Cys codons, which enabled the discovery of unnannotated Cys codon-containing genes. We demonstrate that some are orthologous to small open reading frames (sORFs) that act as Cys-responsive attenuators as recently described in M. smegmatis (15). Ribosome stalling also unleashes a cascade of downstream events that precisely manipulates the transcriptome based on codon usage to down-regulate growth and redirect metabolism toward the synthesis of Cys, which in turn selectively activates genes involved in the oxidative stress and copper detoxification responses, endowing Mtb with the molecular defenses needed to withstand the deadly assaults of the host innate immune response.…”

“…5A) (21). Moreover, recent follow-up studies on the function of Ms5788A support its role in a novel mode of Cys attenuation (15). Ms5788A is so named because it exerts translational control of the downstream three-gene operon beginning with the Ms5788 (unknown function) followed by the M. smegmatis CysA2 sulfotransferase and SseC2 putative sulfotransferase, both of whose up-regulation is logical under Cys-limiting conditions.…”

“…preclude formation of a secondary structure that would otherwise block the availability of the Shine-Dalgarno (SD) ribosome binding site needed to recruit the ribosome and translate the downstream mRNA (Fig. 5B) (15). Distinct from the classic tryptophan attenuation in Escherichia coli, where the absence or presence of ribosome stalling at tryptophan codons in a small leader peptide dictates the formation of alternate secondary structures that either permit or prevent transcription termination, this M. smegmatis Cysrich small protein acts as a sensor for Cys concentration and dictates whether transcripts that follow are translated.…”

“…Based on the function of the Ms5788A sORF in M. smegmatis, we predicted that its leaderless Mtb counterpart in front of an operon with similar architecture would also serve as a Cys-responsive attenuator. We designated this Mtb sORF as Rv0815A, following the convention of newly annotated genes in Mycobrowser [also used by Canestrari et al (15)] to add an A to the name of the gene it precedes, since it resides 173 nt in front of the cysA2 (Rv0815c) and sseC2 genes (Fig. 5A).…”