Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.During protein synthesis, the ribosome catalyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as a template and aminoacylated tRNAs (aatRNAs) as substrates. Correct base pairing between the three bases of the codon on mRNA and those of the anticodon of the cognate aatRNA dictates the sequence of the polypeptide chain. Discrimination against noncognate tRNA, which generally has two or three mismatches in the base pairing, can be accounted for by the difference in the free energy of base pairing to the codon compared with cognate tRNA. For near-cognate tRNA, which usually involves a single mismatch, the free-energy difference in base pairing compared with cognate tRNA would predict an error rate that is one to two orders of magnitude higher than the actual error rate of protein synthesis (1), and it has long been recognized that the ribosome must improve the accuracy of protein synthesis by discriminating against near-cognate tRNAs (2). This discrimination involves the 30S subunit, which binds mRNA and the anticodon stem-loop (ASL) of tRNA.At the beginning of the elongation cycle, which involves the addition of a new amino acid to a growing polypeptide chain, the aatRNA is presented to the ribosome as a ternary complex with elongation factor Tu (EF-Tu) and guanosine triphosphate (GTP). The selection of cognate tRNA is believed to occur in two stages-an initial recognition step and a proofreading step-that are separated by the irreversible hydrolysis of GTP by EF-Tu (3-6). In this scheme, the discrimination energy inherent in codon-anticodon base pairing is exploited twice to achieve the necessary accuracy. Recent experiments suggest that the binding of cognate rather than near-cognate tRNA results in higher rates of both GTP hydrolysis by EF-Tu, and accommodation, a process in which the acceptor arm of the aa-tRNA swings into the peptidyl transferase site after its release from EF-Tu (7,8). In both steps, the higher rate is proposed to be the result of structural changes in the ribosome induced by cognate tRNA. In the context of proofreading mechanisms alone, it is unclear whether additional structural discrimination by the ribosome, ...

A structural and mechanistic explanation for the selection of tRNAs by the ribosome has been elusive. Here, we report crystal structures of the 30S ribosomal subunit with codon and near-cognate tRNA anticodon stem loops bound at the decoding center and compare affinities of equivalent complexes in solution. In ribosomal interactions with near-cognate tRNA, deviation from Watson-Crick geometry results in uncompensated desolvation of hydrogen-bonding partners at the codon-anticodon minor groove. As a result, the transition to a closed form of the 30S induced by cognate tRNA is unfavorable for near-cognate tRNA unless paromomycin induces part of the rearrangement. We conclude that stabilization of a closed 30S conformation is required for tRNA selection, and thereby structurally rationalize much previous data on translational fidelity.

A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used ''Walker strains'' C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for highthroughput applications.engineering ͉ systems biotechnology ͉ proteomics T he natural abundance of membrane proteins is typically too low to isolate sufficient amounts of material for functional and structural studies. Therefore, membrane proteins must be obtained by overexpression, and the bacterium E. coli is the most widely used vehicle for this purpose (1). Although many membrane proteins can be overexpressed in inclusion bodies, their refolding into functional proteins is often not successful (2). To avoid the refolding problem, overexpression of membrane proteins by accumulation in the cytoplasmic membrane is needed. However, overexpression is often toxic to the cell, thereby preventing biomass formation and severely reducing yields (1). Thus, membrane protein overexpression has to be optimized, but no systematic, generic, and high-throughput-compatible method is available for the optimization process.Bacteriophage T7 RNA polymerase (T7RNAP) is often used to drive recombinant protein production in E. coli (3). In BL21(DE3) and its derivatives, the gene encoding T7RNAP is under control of the lacUV5 promoter, a strong variant of the wild-type lac promoter. It is insensitive to catabolite repression and, therefore, controlled only by the lac repressor, LacI, which binds to the lac operator (4). T7RNAP exclusively recognizes the T7 promoter and it transcribes eight times faster than E. coli RNAP allowing high yield protein production (5). Most T7 expression vectors employ a T7lac hybrid promoter that combines the strong T7 10 promoter with a lac operator to diminish leaky expression. On addition of the inducer isopropyl ␤-Dthiogalactoside (IPTG), lacI repression is relieved, resulting in recombinant protein production. If toxicity due to leaky expression is a problem, T7RNAP activity can be further dampened with the T7RNAP inhibit...

The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.

During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.