Autogenous regulation is a general strategy of balancing ribosomal protein synthesis in bacteria. Control mechanisms have been studied in detail for most of ribosomal protein operons, except for rpsB-tsf encoding essential r-protein S2 and elongation factor Ts, where even the promoter has remained unknown. By using single-copy translational fusions with the chromosomal lacZ gene and Western-blot analysis, we demonstrate here that S2 serves as a negative regulator of both rpsB and tsf expression in vivo, acting at a single target within the rpsB 5′-untranslated region (5′-UTR). As determined by primer extension, transcription of the Escherichia coli rpsB-tsf operon starts 162 nucleotides upstream of the rpsB initiation codon at a single promoter TGTGGTATAAA belonging to the extended -10 promoter class. Both the promoter signature and the 5′-UTR structure of the rpsB gene appear to be highly conserved in γ-proteobacteria. Deletion analysis of the rpsB 5′-UTR within rpsB′-′lacZ fusions has revealed that an operator region involved in the S2 autoregulation comprises conserved structural elements located upstream of the rpsB ribosome binding site. The S2-mediated autogenous control is impaired in rpsB mutants and, more surprisingly, in the rpsA mutant producing decreased amounts of truncated r-protein S1 (rpsA::IS10), indicating that S2 might act as a repressor in cooperation with S1. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.