ABSTRACT
Genome-wide transcriptomic analyses have revealed abundant expressed short open reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins they encode, are functional remains an open question. One quarter of mycobacterial mRNAs are leaderless, meaning the RNAs begin with a 5’-AUG or GUG initiation codon. Leaderless mRNAs often encode an unannotated short ORF as the first gene of a polycistronic transcript. Consecutive cysteine codons are highly overrepresented in mycobacterial leaderless short ORFs. Here we show that polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression. Through detailed mutational analysis, we show that one such polycysteine-encoding short ORF controls expression of the downstream genes by causing ribosome stalling under conditions of low cysteine. Ribosome stalling in turn blocks mRNA secondary structures that otherwise sequester the Shine-Dalgarno ribosome-binding site of the 3’gene. This translational attenuation does not require competing transcriptional terminator formation, a mechanism that underlies traditional amino acid attenuation systems. We further assessed cysteine attenuation in Mycobacterium smegmatis using mass spectrometry to evaluate endogenous proteomic responses. Notably, six cysteine metabolic loci that have unannotated polycisteine-encoding leaderless short ORF architectures responded to cysteine supplementation/limitation, indicating that cysteine-responsive attenuation is widespread in mycobacteria. Individual leaderless short ORFs confer independent operon-level control, while their shared dependence on cysteine ensures a collective response. Bottom-up regulon coordination is the antithesis of traditional top-down master regulator regulons and illustrates one utility of the many unnanotated short ORFs expressed in bacterial genomes.
