Role of conserved surface amino acids in binding of SmpB protein to SsrA RNA.
Bacteria possess a unique salvage mechanism for rescuing ribosomes stalled on aberrant mRNAs. A complex of SmpB protein and SsrA RNA orchestrates this salvage process. The specific and direct binding of SmpB facilitates recognition and delivery of SsrA RNA to stalled ribosomes. The SmpB protein is conserved throughout the bacterial kingdom and contains several conserved amino acid sequence motifs. We present evidence to demonstrate that amino acid residues Glu-31, Leu-91, and Lys-124, which are highly conserved and clustered along an exposed surface of the protein, play a crucial role in the SsrA-mediated peptide tagging process. Our analysis suggests that the peptide-tagging defect exhibited by these SmpB variants is due to their inability to facilitate the delivery of SsrA RNA to stalled ribosomes. Moreover, we present evidence to demonstrate that the ribosome association defect of these variants is due to their reduced SsrA binding affinity. Consistent with these findings, we present biochemical evidence to demonstrate that residues Glu-31, Leu-91, and Lys-124 are essential for the SsrA binding activity of SmpB protein. Furthermore, we have investigated the interactions of SmpB.SsrA orthologues from the thermophilic bacterium Thermoanaerobacter tengcongensis. Our investigations demonstrate an analogous role for the equivalent T. tengcongensis residues in SmpB.SsrA interactions, hence further validating our findings for the Escherichia coli SmpB.SsrA system. These results demonstrate the functional significance of this cluster of conserved residues in SmpB binding to SsrA RNA, suggesting they might represent a core contact surface for recognition of SsrA RNA.
Dulebohn
Daniel P
DP
Department of Biochemistry and Cell Biology and Center for Infectious Diseases of Stony Brook University, Stony Brook, New York 11794, USA.
Cho
Hye Jin
HJ
Karzai
A Wali
AW
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D017124:Conserved Sequence; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry; D046968:Thermoanaerobacter / Q000378:metabolism
D000596:Amino Acids; D010455:Peptides; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA; D012313:RNA