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...

New developments concerning alignment media for apolar solvents like chloroform make it possible to measure anisotropic parameters such as residual dipolar couplings (RDCs) at relatively low concentrations and natural isotopic abundance. As RDCs provide structural restraints with respect to an external coordinate system, long-range structural arrangements of the time-averaged structure can be determined with high precision. The method is demonstrated on the well-studied cyclo-undecapeptide Cyclosporin A (CsA), for which crystal and conventionally derived NMR structures are available. Neither crystal nor NMR structure are consistent with heteronuclear D(CH) RDCs measured in a stretched poly(dimethylsiloxane) gel, and refinement by using the anisotropic parameter results in a highly defined structure with a slightly changed backbone conformation. The applied methods and interpretation of the structural model are discussed.

No abstract

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1–2‰ for sulfate δ34S and δ18O and nitrate δ15N and δ18O, based on in-house and international standards. Less abundant variants such as δ17O, δ33S, and δ36S, and the 34S–18O “clumped” sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine 18O/34S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

No abstract

Our ability to read the molecular fossil record has advanced significantly in the past decade. Improvements in biomarker sampling and quantification methods, expansion of molecular sequence databases, and the application of genetic and cellular biological tools to problems in biomarker research have enabled much of this progress. By way of example, we review how attempts to understand the biological function of 2-methylhopanoids in modern bacteria have changed our interpretation of what their molecular fossils tell us about the early history of life. They were once thought to be biomarkers of cyanobacteria and hence the evolution of oxygenic photosynthesis, but we now believe that 2-methylhopanoid biosynthetic capacity originated in the Alphaproteobacteria, that 2-methylhopanoids are regulated in response to stress, and that hopanoid 2-methylation enhances membrane rigidity. We present a new interpretation of 2-methylhopanes that bridges the gap between studies of the functions of 2-methylhopanoids and their patterns of occurrence in the rock record.