The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release.
SummaryTranslational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 Å) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 Å) and after (3.6 Å) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2′-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.
In bacteria, ribosomes stalled at the end of truncated messages are rescued by tmRNA, a bifunctional molecule that acts as both a tRNA and mRNA, and SmpB, a small protein that works in concert with tmRNA. Here we present the crystal structure at 3.2 Å resolution of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome. The structure shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome, and explains why the tmRNASmpB system does not interfere with normal translation.Transfer-messenger RNA (tmRNA), also known as 10S RNA or SsrA, is a highly structured RNA that combines properties of tRNA and mRNA in one molecule about 350 nucleotides long (Fig. 1A) (1, 2). The tRNA-like domain (TLD) of tmRNA lacks an anticodon stem loop but contains an acceptor arm (3) that can be aminoacylated at its 3′-end by the same alanyl tRNA synthetase that aminoacylates tRNA Ala . A different region of tmRNA contains a short internal open reading frame (ORF) that acts as an mRNA template. In addition, tmRNA contains several pseudoknots and helices.Ribosomes that reach the end of prematurely truncated or defective messages are stalled because the absence of a complete codon in the A site prevents either elongation or normal termination. In bacteria, they are rescued by tmRNA in a process called trans-translation because it involves continuing translation by changing the mRNA template. In this process, EF-Tu delivers tmRNA to the A site of the stalled ribosome. The nascent polypeptide chain is transferred to the alanine on the TLD. Subsequently, translocation brings the first codon of the ORF into the A site of the ribosome and translation resumes using the ORF as the mRNA (2). The short sequence coded by the ORF thus added to the C-terminus of the partially synthesized protein acts as a degradation tag (4). Thus tmRNA acts both to rescue ribosomes as well as to target incompletely synthesized proteins for degradation.The binding of tmRNA to stalled ribosomes requires the protein SmpB (5), which can bind to tmRNA simultaneously with EF-Tu (6). Crystal structures of SmpB in complex with the TLD suggest that the protein substitutes for the missing anticodon stem inside the ribosome (7, 8), which was supported by electron microscopy studies at ~15 Å resolution (9, 10). A previous electron microscopy study found two molecules of SmpB with tmRNA in the ribosome, with the carboxy-terminus of one of them near the decoding center of the 30S ribosomal subunit (11). The observed proximity to the decoding center agrees with hydroxyl * To whom correspondence should be addressed at ramak@mrc-lmb. (14). The mechanism by which tmRNA and SmpB acting in concert can facilitate "decoding" in the absence of codon-anticodon base pairing has remained unclear.Here we present the crystal structure of the Thermus thermophilus ribosome bound to a complex consisting of a fragment of tmRNA (tmRNAΔ m ) along with SmpB and EF-Tu t...
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