Kurt Fredrick

How the sequence of a gene can tune its translation.

Sixty-one codons specify 20 amino acids, offering cells many options for encoding a polypeptide sequence. Two new studies (Cannarrozzi et al., 2010; Tuller et al., 2010) now foster the idea that patterns of codon usage can control ribosome speed, fine-tuning translation to increase the efficiency of protein synthesis.

A steric block in translation caused by the antibiotic spectinomycin.

The widely used antibiotic spectinomycin inhibits bacterial protein synthesis by blocking translocation of messenger RNA and transfer RNAs on the ribosome. Here, we show that in crystals of the Escherichia coli 70S ribosome spectinomycin binding traps a distinct swiveling state of the head domain of the small ribosomal subunit. Spectinomycin also alters the rate and completeness of reverse translocation in vitro. These structural and biochemical data indicate that in solution spectinomycin sterically blocks swiveling of the head domain of the small ribosomal subunit and thereby disrupts the translocation cycle.

Ribosomal Translocation: One Step Closer to the Molecular Mechanism

Protein synthesis occurs in ribosomes, the targets of numerous antibiotics. How these large and complex machines read and move along mRNA have proven to be challenging questions. In this Review, we focus on translocation, the last step of the elongation cycle in which movement of tRNA and mRNA is catalyzed by elongation factor G. Translocation entails large-scale movements of the tRNAs and conformational changes in the ribosome that require numerous tertiary contacts to be disrupted and reformed. We highlight recent progress toward elucidating the molecular basis of translocation and how various antibiotics influence tRNA-mRNA movement.

Structural basis for hygromycin B inhibition of protein biosynthesis

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.

Role of hybrid tRNA-binding states in ribosomal translocation

During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steady-state kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (ktrans), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K1/2). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K1/2 conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases ktrans without increasing K1/2. These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.

Resampling and Editing of Mischarged tRNA Prior to Translation Elongation

Faithful translation of the genetic code depends on the GTPase EF-Tu delivering correctly charged aminoacyl-tRNAs to the ribosome for pairing with cognate codons. The accurate coupling of cognate amino acids and tRNAs by the aminoacyl-tRNA synthetases is achieved through a combination of substrate specificity and product editing. Once released by aminoacyl-tRNA synthetases, both cognate and near-cognate aminoacyl-tRNAs were considered to be committed to ribosomal protein synthesis through their association with EF-Tu. Here we show instead that aminoacyl-tRNAs in ternary complex with EF-Tu*GTP can readily dissociate and rebind to aminoacyl-tRNA synthetases. For mischarged species, this allows resampling by the product editing pathway, leading to a reduction in the overall error rate of aminoacyl-tRNA synthesis. Resampling of mischarged tRNAs was shown to increase the accuracy of translation over ten fold during in vitro protein synthesis, supporting the presence of an additional quality control step prior to translation elongation.

Translation factor LepA contributes to tellurite resistance in Escherichia coli but plays no apparent role in the fidelity of protein synthesis

LepA is a translational GTPase highly conserved in bacterial lineages. While it has been shown that LepA can catalyze reverse ribosomal translocation in vitro, the role of LepA in the cell remains unclear. Here, we show that deletion of the lepA gene (DeltalepA) in Escherichia coli causes hypersensitivity to potassium tellurite and penicillin G, but has no appreciable effect on growth under many other conditions. DeltalepA does not increase miscoding or frameshifting errors under normal or stress conditions, indicating that LepA does not contribute to the fidelity of translation. Overexpression of LepA interferes with tmRNA-mediated peptide tagging and A-site mRNA cleavage, suggesting that LepA is a bona fide translation factor that can act on stalled ribosomes with a vacant A site in vivo. Together these results lead us to hypothesize that LepA is involved in co-translational folding of proteins that are otherwise vulnerable to tellurite oxidation.

Preparation and evaluation of acylated tRNAs

In the cell, the activity of tRNA is governed by its acylation state. Interactions with the ribosome, translation factors, and regulatory elements are strongly influenced by the acyl group, and presumably other cellular components that interact with tRNA also use the acyl group as a specificity determinant. Thus, those using biochemical approaches to study any aspect of tRNA biology should be familiar with effective methods to prepare and evaluate acylated tRNA reagents. Here, methods to prepare aminoacyl-tRNA, N-acetyl-aminoacyl-tRNA, and fMet-tRNA(fMet) and to assess their homogeneity are described. Using these methods, acylated tRNAs of high homogeneity can be reliably obtained.

Paternal chromosome segregation during the first mitotic division determines Wolbachia-induced cytoplasmic incompatibility phenotype.

The most common Wolbachia-induced phenotype in insects is cytoplasmic incompatibility (CI), which occurs when sperm from infected males fertilize eggs from uninfected females. CI produces distinct phenotypes in three closely related haplo-diploid species of the genus Nasonia: mortality in N. longicornis and N. giraulti, and conversion to male development in N. vitripennis. We demonstrate that the majority of CI-induced mortality occurs during embryogenesis and that the pattern of paternal chromosome segregation during the first mitosis is a good predictor of CI phenotype. In N. giraulti and N. longicornis, the paternal chromosomes mis-segregate, producing abnormal nuclei connected by chromatin bridges. Consequently, these embryos arrest development with very few and abnormal nuclei. In contrast, the paternal genome in N. vitripennis is either not segregated or mis-segregates to one of the two daughter nuclei. Consequently, these embryos continue development utilizing the maternally derived haploid nuclei, resulting in male offspring. The latter class is the first documented example of asymmetric mitotic segregation of abnormal chromosomes. We conclude that in haplo-diploids, CI-induced embryonic lethality occurs only when abnormal paternal genome segregation affects both products of the first mitotic division. This is the first study to associate differences in types of CI with specific cytological defects.

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection.