Contributions of pseudoknots and protein SmpB to the structure and function of tmRNA in trans-translation.
Bacteria contain transfer-messenger RNA (tmRNA), a molecule that during trans-translation tags incompletely translated proteins with a small peptide to signal the proteolytic destruction of defective polypeptides. TmRNA is composed of tRNA- and mRNA-like domains connected by several pseudoknots. Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli tmRNA in trans-translation. Site-directed mutagenesis experiments showed that pseudoknot 2 and the abutting helix 5 were particularly important for the binding of ribosomal protein S1 to tmRNA. Pseudoknot 4 not only facilitated tmRNA maturation but also promoted tagging. In addition, the three pseudoknots (pk2 to pk4) were shown to play a significant role in the proper folding of the tRNA-like domain. Protein SmpB enhanced tmRNA processing, suggesting a new role for SmpB in trans-translation. Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis.
Wower
Iwona K
IK
Department of Animal Sciences, Auburn University, Auburn, Alabama 36849-5415, USA. iwower@acesag.auburn.edu
Zwieb
Christian
C
Wower
Jacek
J
D020029:Base Pairing; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C015494:ribosomal protein S1; C430328:small protein B; C096222:tmRNA; D012343:RNA, Transfer