Surprising flexibility of leader RNA determinants for r-protein L4-mediated transcription termination in the Escherichia coli S10 operon

Abstract

Escherichia coli ribosomal protein L4 autogenously regulates transcription of the S10 operon, which encodes L4 and 10 other ribosomal proteins. Regulation results from L4-stimulated premature transcription termination at a U-rich site in the untranslated leader. The process requires transcription factor NusA. Here we report a detailed analysis of the RNA requirements for NusA-dependent, L4-mediated transcription control. We found that efficient regulation requires multiple features of the S10 leader, including two hairpins, called HD and upper HE, a connecting tether, and a U-rich sequence at the distal side of HE. As expected, regulation was optimal when all 7 Us were maintained in the U₄CGU₃ sequence at the termination site. However, despite the apparent specificity of L4 action on only the S10 operon, there is surprising flexibility at the primary sequence level for the HD-tether-HE region. Changes in the sequence of non-base-paired nucleotides flanking the HD hairpin or an A at the second position of the HD loop reduced L4 regulation, but other changes had little or no effect. Furthermore, generic hairpins from other RNAs could replace the natural HD and upper HE hairpins with little or no reduction of L4 control, suggesting that the secondary structure elements are also relatively generic. The lack of specific sequence requirements suggests that L4 may recognize multiple elements within this region of the nascent leader.