anticodon and mocimycin

Topics: Anticodon; Codon; Codon, Terminator; Cryoelectron Microscopy; Nucleic Acid Conformation; Open Reading Frames; Peptide Elongation Factor Tu; Protein Biosynthesis; Pyridones; Ribosomes; RNA-Binding Proteins; RNA, Bacterial; RNA, Messenger; RNA, Transfer; Thermus thermophilus

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.

Topics: Alanine; Anticodon; Bacterial Proteins; Codon; Codon, Terminator; Cryoelectron Microscopy; Guanosine Triphosphate; Image Processing, Computer-Assisted; Models, Molecular; Nucleic Acid Conformation; Open Reading Frames; Peptide Elongation Factor Tu; Protein Biosynthesis; Pyridones; Ribosomal Proteins; Ribosomes; RNA-Binding Proteins; RNA, Bacterial; RNA, Messenger; RNA, Transfer; Thermus thermophilus

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Topics: Anticodon; Codon; Cryoelectron Microscopy; Escherichia coli; Escherichia coli Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Image Processing, Computer-Assisted; Macromolecular Substances; Models, Molecular; Nucleic Acid Conformation; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; Ribosomes; RNA, Transfer; RNA, Transfer, Amino Acyl; RNA, Transfer, Phe; Structure-Activity Relationship

The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.

Topics: Anticodon; Binding Sites; Codon; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Nucleic Acid Conformation; ortho-Aminobenzoates; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl