In eubacteria, the rpsB-tsf operon encodes two essential components of the translational apparatus, ribosomal protein S2 and elongation factor Ts. We have recently localized the promoter region of the Escherichia coli rpsB-tsf operon and have demonstrated that both rpsB and tsf are negatively regulated by S2 at the translational level. In this work, phylogenetic analysis showed high conservation of both the promoter signature and the structure of the 5?-untranslated region (5?-UTR) of the rpsB mRNA in γ-proteobacteria. Despite the difference in length and overall primary structure of rpsB 5?-UTRs among various γ-proteobacteria, several short regions within 5?-UTRs proved to be universally conserved, implying their participation in expression regulation. Phylogenetic predictions were experimentally verified. The putative rpsB promoter regions of Yersinia pestis, Haemophilus influenzae, and Pseudomonas aeruginosa drove transcription of the lacZ reporter in E. coli, and the corresponding rpsB 5?-UTRs were subject to autogenous repression by S2 in vivo. © 2009 Pleiades Publishing, Ltd.