An S6:S18 complex inhibits translation of E. coli rpsF

• Published in: RNA (Cambridge) 2015-12, Vol.21 (12), p.2039-2046
• Main Authors: Babina, Arianne M, Soo, Mark W, Fu, Yang, Meyer, Michelle M
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - biosynthesis; Escherichia coli Proteins - genetics; Gene Expression Regulation, Bacterial; gene regulation; Protein Biosynthesis; Regulatory Sequences, Ribonucleic Acid; Ribosomal Proteins - biosynthesis; Ribosomal Proteins - genetics; ribosome; RNA cis-regulator; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; structured RNA; translation inhibition
• ISSN: 1355-8382; 1469-9001; 1469-9001
• DOI: 10.1261/rna.049544.115

More than half of the ribosomal protein operons in Escherichia coli are regulated by structures within the mRNA transcripts that interact with specific ribosomal proteins to inhibit further protein expression. This regulation is accomplished using a variety of mechanisms and the RNA structures responsible for regulation are often not conserved across bacterial phyla. A widely conserved mRNA structure preceding the ribosomal protein operon containing rpsF and rpsR (encoding S6 and S18) was recently identified through comparative genomics. Examples of this RNA from both E. coli and Bacillus subtilis were shown to interact in vitro with an S6:S18 complex. In this work, we demonstrate that in E. coli, this RNA structure regulates gene expression in response to the S6:S18 complex. β-galactosidase activity from a lacZ reporter translationally fused to the 5' UTR and first nine codons of E. coli rpsF is reduced fourfold by overexpression of a genomic fragment encoding both S6 and S18 but not by overexpression of either protein individually. Mutations to the mRNA structure, as well as to the RNA-binding site of S18 and the S6-S18 interaction surfaces of S6 and S18, are sufficient to derepress β-galactosidase activity, indicating that the S6:S18 complex is the biologically active effector. Measurement of transcript levels shows that although reporter levels do not change upon protein overexpression, levels of the native transcript are reduced fourfold, suggesting that the mRNA regulator prevents translation and this effect is amplified on the native transcript by other mechanisms.