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.

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编码多半胱氨酸的无前导短 ORF 可作为分枝杆菌中操纵基因表达的半胱氨酸响应衰减器。

全基因组转录组分析揭示了细菌中大量表达的短开放阅读框（ORF）。这些短 ORF 或它们编码的小蛋白质是否具有功能仍然是一个悬而未决的问题。四分之一的分枝杆菌 mRNA 是无前导的，以 5'-AUG 或 GUG 起始密码子开始。无前导 mRNA 通常将未注释的短 ORF 编码为多顺反子转录本的第一个基因。在这里，我们展示了编码多半胱氨酸的无前导短 ORF 可作为半胱氨酸响应性抑制基因表达。详细的突变分析表明，一个多半胱氨酸短 ORF 控制下游基因的表达。我们的数据表明，在多半胱氨酸束中停滞的核糖体阻断了 mRNA 结构，否则这些结构会隔离 3' 基因的核糖体结合位点。使用质谱法的耻垢分枝杆菌。六个半胱氨酸代谢位点具有未注释的编码多半胱氨酸的无前导短 ORF 结构对半胱氨酸限制有反应，揭示了分枝杆菌中广泛存在的半胱氨酸反应衰减。单个无领导的短 ORF 赋予独立的操纵子水平控制，而它们对半胱氨酸的共同依赖确保了由核糖体暂停介导的集体反应。我们提出术语 ribulon 来分类核糖体导向的调节子。核糖体在感觉短 ORF 上的调节子水平协调说明了细菌基因组中表达的许多未注释的短 ORF 的一种用途。