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Polycysteine‐encoding leaderless short ORFs function as cysteine‐responsive attenuators of operonic gene expression in mycobacteria
Author(s) -
Canestrari Jill G.,
LasekNesselquist Erica,
Upadhyay Ashutosh,
Rofaeil Martina,
Champion Matthew M.,
Wade Joseph T.,
Derbyshire Keith M.,
Gray Todd A.
Publication year - 2020
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.14498
Subject(s) - orfs , biology , regulon , open reading frame , genetics , start codon , gene , operon , mycobacterium smegmatis , bacterial genome size , genome , gene expression , messenger rna , peptide sequence , escherichia coli , mycobacterium tuberculosis , medicine , tuberculosis , pathology
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, beginning with a 5′‐AUG or GUG initiation codon. Leaderless mRNAs often encode unannotated short ORFs as the first gene of a polycistronic transcript. Here, we show that polycysteine‐encoding leaderless short ORFs function as cysteine‐responsive attenuators of operonic gene expression. Detailed mutational analysis shows that one polycysteine short ORF controls expression of the downstream genes. Our data indicate that ribosomes stalled in the polycysteine tract block mRNA structures that otherwise sequester the ribosome‐binding site of the 3′gene. We assessed endogenous proteomic responses to cysteine limitation in Mycobacterium smegmatis using mass spectrometry. Six cysteine metabolic loci having unannotated polycysteine‐encoding leaderless short ORF architectures responded to cysteine limitation, revealing widespread cysteine‐responsive attenuation in mycobacteria. Individual leaderless short ORFs confer independent operon‐level control, while their shared dependence on cysteine ensures a collective response mediated by ribosome pausing. We propose the term ribulon to classify ribosome‐directed regulons. Regulon‐level coordination by ribosomes on sensory short ORFs illustrates one utility of the many unannotated short ORFs expressed in bacterial genomes.