Translational errors as the cause of mutations in Escherichia coli

Summary The present work suggests that a significant proportion of spontaneous mutations in Escherichia coli are the result of translational errors. This idea is supported by the following observations: (i) Streptomycin can induce the formation of auxotrophic mutants in streptomycin-sensitive cells, but not in rpsL mutants resistant to streptomycin, and (ii) strains having hyper-accurate ribosomes (rpsL999 and rpsL1204 strains) show reduced mutation rates. The implications of these results are discussed with respect to the dogma of randomness of spontaneous mutations and the directed mutation hypothesis.     

Erythromycin, lincosamides, peptidyl-tRNA dissociation, and ribosome editing

Inaccurate protein synthesis produces unstable -galactosidase, whose activity is rapidly lost at high temperature. Erythromycin, lincomycin, clindamycin, and celesticetin were shown to counteract the error-inducing effects of streptomycin on -galactosidase synthesized in the antibiotic-hypersensitive Escherichia coli strain DB-11 Met ^–. Newly synthesized -galactosidase was more easily inactivated by high temperatures when synthesized by bacteria partially starved for arginine, threonine, or methionine. Simultaneous treatment with erythromycin or linocomycin yielded -galactosidase that was inactivated by high temperatures less easily than during starvation alone, an effect attributed to stimulation of ribosome editing. When synthesized in the presence of canavanine, -galactosidase was inactivated by high temperature more easily but this effect could not be reversed by erythromycin. The first arginine in -galactosidase occurs at residue 13, so the effect of erythromycin during arginine starvation is probably to stimulate dissociation of erroneous peptidyl-tRNAs of at least that length. Correction of errors induced by methionine starvation is probably due to stimulation of dissociation of erroneous peptidyl-tRNAs bearing peptides at least 92 residues in length. All the effects of erythromycin or the tested lincosamides on protein synthesis are probably the result of stimulating the dissociation from ribosomes of peptidyl-tRNAs that are erroneous or short.     

Recognition and selection of tRNA in translation

Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. The stepwise movement of aa-tRNA from EF-Tu into the ribosomal A site entails a number of intermediates. The ribosome recognizes aa-tRNA through shape discrimination of the codon-anticodon duplex and regulates the rates of GTP hydrolysis by EF-Tu and aa-tRNA accommodation in the A site by an induced fit mechanism. Recent results of kinetic measurements, ribosome crystallography, single molecule FRET measurements, and cryo-electron microscopy suggest the mechanism of tRNA recognition and selection.     

Engine out of the chassis: Cell-free protein synthesis and its uses

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching to tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression.     

Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.     

Estimating the sensitivity of cervical cytology: errors of interpretation and test limitations

The objective of this study was to estimate: (i) the sensitivity of cytologists in recognizing abnormal smears; (ii) the sensitivity of cervical cytology as a method of detecting abnormal smears among those obtained in the presence of cervical intraepithelial neoplasia (CIN). Study subjects were 61 women with a histologically confirmed CIN identified through colpohistological and cytologic screening. For objective (i) new smears were taken from study subjects just before treatment, mixed with routine preparations, interpreted by unaware cytologists and then blindly reviewed by a group of three expert supervisors, who reached a consensus diagnosis. Cytologists classified as positive for squamous intraepithelial lesion (SIL) 30 of the 34 smears judged as positive by supervisors (100% of smears classified as high-grade and 67% of smears classified as low-grade SIL by the supervisors). Our approach, based on creating a set of smears with a high a priori probability of being positive, proved to be an efficient way of estimating errors of interpretation. For objective (ii), smears taken at the moment of diagnosis, just before biopsy, were also reviewed by the same supervisors. These CIN cases were identified among asymptomatic women independently of cytological findings and results are therefore not subject to verification bias. Among the 33 histological CINII/III, four (12%) smears had no atypical cells (three negatives and one unsatisfactory) at review. The same proportion was 26% (four negatives and one unsatisfactory) among the 19 histological CINI. No significant differences in smear content were found between the seven ‘false negatives’ and a sample of ‘true positives’ and ‘true negatives’ for a number of formal adequacy criteria (including presence of endocervical cells). Strong differences were found between positive smears taken just before biopsy and those taken just before treatment (in 11 women the first smear only was positive, while the opposite was never observed), suggesting an effect of punch biopsy in removing lesions.     

Biphasic Modulation by ACTH-Like Peptides of Protein Synthesis in a Cell-Free System from Rat Brain

Brain protein synthesis in a cell-free system was stimulated by 10(-8) M-ACTH1-24. This stimulatory effect was completely inhibited by aurintricarboxylic acid (ATA), an inhibitor of reinitiation of new peptide chains. The N-terminal peptide sequence 4-10 exerted a biphasic modulation of cell-free protein synthesis, i.e., a stimulation at low concentrations (10(-8) and 10(-10) M) and an inhibition at a high concentration (10(-4) M). The D-isomer, ACTH4-10-7-D-phe, also showed a biphasic modulation that, however, was in a direction opposite to that shown by ACTH4-10-7-L-phe at 10(-8) M and 10(-4) M.     

Possible role for protein kinase C in the pathogenesis of inborn errors of metabolism

Protein kinase C (PKC) is a ubiquitous enzyme family implicated in the regulation of a large number of short- and long-term intracellular processes. It is hypothesized that modulation of PKC activity may represent, at least in part, a functional link between mutations (genotype) that lead to the pathological accumulation of naturally occurring compounds that affect PKC activity and perturbation of PKC-mediated substrate phosphorylation and cellular function in the corresponding diseases (phenotype). This model provides a unifying putative mechanism by which the phenotypic expression of some inborn errors of metabolism may be explained. Recent studies in a cell-free system of human skin fibroblasts support the hypothesis that alteration of PKC activity may represent the functional link between accumulation of sphingolipids and fatty acyl-CoA esters, and perturbation of cell function in sphingolipidoses and fatty acid oxidation defects, respectively. Further studies will elucidate the effects of these alterations on PKC-mediated short- and long-term cellular functions in these diseases, as well as the possible role of PKC in the pathogensis of other diseases. © 1995 Wiley-Liss, Inc.     

Rate, accuracy and cost of ribosomes in bacterial cells

Recent biochemical data on the rate of peptidyl-transfer and missense error levels associated with the E. coli ribosome in conjunction with direct measurements of diffusion constants for proteins in the E. coli cell have been used to discuss protein synthesis in the living E. coli cell in the perspective of a previously developed maximal fitness theory. With these improved experimental parameters, i.e. kcat approximately 50 s(-1) for protein elongation and kcat/KM approximately 4 microM(-1) s(-1) for cognate ternary complex binding to the ribosomal A site, theory predicts the experimentally observed variations in protein elongation rate, ribosome and ternary complex concentrations with varying quality of the growth medium. The theoretically predicted average missense error level is close the error levels estimated in vitro for special isoacceptor combinations, i.e. error levels about 1 per million. The future prospect of extensive integration of biochemistry, cell physiology and population genetics is discussed in the light of the maximal fitness theory and other, similar, theoretical approaches.     

Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues

A method for the rapid generation of intact proteins in a cell-free protein synthesis system was developed. The productivity of the recombinant proteins from the polymerase-chain-reaction-amplified templates was enhanced remarkably using an optimized translation enhancer sequence. The extra amino acid residues derived from the translation enhancer sequence were effectively removed by utilizing the appropriate detergent and peptide cleavage enzyme in the reaction mixture. These results demonstrate the versatility of cell-free protein synthesis in providing optimized and customized reaction conditions for the efficient production of the desired proteins.     

Sequence-structure mapping errors in the PDB: OB-fold domains

The Protein Data Bank (PDB) is the single most important repository of structural data for proteins and other biologically relevant molecules. Therefore, it is critically important to keep the PDB data, as much as possible, error-free. In this study, we have analyzed PDB crystal structures possessing oligonucleotide/oligosaccharide binding (OB)-fold, one of the highly populated folds, for the presence of sequence-structure mapping errors. Using energy-based structure quality assessment coupled with sequence analyses, we have found that there are at least five OB-structures in the PDB that have regions where sequences have been incorrectly mapped onto the structure. We have demonstrated that the combination of these computation techniques is effective not only in detecting sequence-structure mapping errors, but also in providing guidance to correct them. Namely, we have used results of computational analysis to direct a revision of X-ray data for one of the PDB entries containing a fairly inconspicuous sequence-structure mapping error. The revised structure has been deposited with the PDB. We suggest use of computational energy assessment and sequence analysis techniques to facilitate structure determination when homologs having known structure are available to use as a reference. Such computational analysis may be useful in either guiding the sequence-structure assignment process or verifying the sequence mapping within poorly defined regions.     

A database for inborn errors of metabolism in the Indian state of Andhra Pradesh

A Database for Inborn Errors of Metabolism (IEM) in the Indian State of Andhra Pradesh (DIEMISAP), is a continuously updated literature depository containing the extensive information on the regional prevalence and heterogeneity of Inborn Errors of metabolism. We report the construction of a database, a flat file and secondary data resource developed using Microsoft Front Page as the core engine. The database contains 18 summaries regarding 43 IEM disorders reported in the AP population, both in general and region wise, with references and links to the IEM disorder databases available on web and institutes involved in IEM research, in India. Summaries can be accessed from the reference given against an alphabetically arranged list of IEM disorders. The DIEMISAP is a useful user friendly and extendable online resource for information on prevalence of IEM in AP. AVAILABILITY: http://biochem.uohyd.ernet.in.     

Further in vitro exploration fails to support the allosteric three-site model

Ongoing debate in the ribosome field has focused on the role of bound E-site tRNA and the Shine-Dalgarno-anti-Shine-Dalgarno (SD-aSD) interaction on A-site tRNA interactions and the fidelity of tRNA selection. Here we use an in vitro reconstituted Escherichia coli translation system to explore the reported effects of E-site-bound tRNA and SD-aSD interactions on tRNA selection events and find no evidence for allosteric coupling. A large set of experiments exploring the role of the E-site tRNA in miscoding failed to recapitulate the observations of earlier studies (Di Giacco, V., Márquez, V., Qin, Y., Pech, M., Triana-Alonso, F. J., Wilson, D. N., and Nierhaus, K. H. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 10715-10720 and Geigenmüller, U., and Nierhaus, K. H. (1990) EMBO J. 9, 4527-4533); the frequency of miscoding was unaffected by the presence of E-site-bound cognate tRNA. Moreover, our data provide clear evidence that the reported effects of the SD-aSD interaction on fidelity can be attributed to the binding of ribosomes to an unanticipated site on the mRNA (in the absence of the SD sequence) that provides a cognate pairing codon leading naturally to incorporation of the purported "noncognate" amino acid.     

Likelihood-based genetic mark-recapture estimates when genotype samples are incomplete and contain typing errors

Genotypes produced from samples collected non-invasively in harsh field conditions often lack the full complement of data from the selected microsatellite loci. The application to genetic mark-recapture methodology in wildlife species can therefore be prone to misidentifications leading to both ‘true non-recaptures’ being falsely accepted as recaptures (Type I errors) and ‘true recaptures’ being undetected (Type II errors). Here we present a new likelihood method that allows every pairwise genotype comparison to be evaluated independently. We apply this method to determine the total number of recaptures by estimating and optimising the balance between Type I errors and Type II errors. We show through simulation that the standard error of recapture estimates can be minimised through our algorithms. Interestingly, the precision of our recapture estimates actually improved when we included individuals with missing genotypes, as this increased the number of pairwise comparisons potentially uncovering more recaptures. Simulations suggest that the method is tolerant to per locus error rates of up to 5% per locus and can theoretically work in datasets with as little as 60% of loci genotyped. Our methods can be implemented in datasets where standard mismatch analyses fail to distinguish recaptures. Finally, we show that by assigning a low Type I error rate to our matching algorithms we can generate a dataset of individuals of known capture histories that is suitable for the downstream analysis with traditional mark-recapture methods.     

The addition of 2,2,2‐trifluoroethanol prevents the aggregation of guanidinium around protein and impairs its denaturation ability: A molecular dynamics simulation study

TFE, 2,2,2‐trifluoroethanol, and guanidinium (Gdm⁺) are two typical osmolytes. A great number of experimental and theoretical studies have shown that TFE and Gdm⁺ can accumulate on protein surface and thus exert their effects on protein structure in their respective solutions. Their accumulation manners are, however, different: the hydrophobic property of TFE makes its accumulation more preferential around hydrophobic side chains than other types whereas Gdm⁺ prefers to stack strongly against the planar side chains but only weakly binds to the hydrophobic groups. The present molecular dynamics simulation study shows a novel test to investigate the combined effects of TFE and Gdm⁺ on protein structure in mixed guanidinium/TFE solution. The results indicate that the accumulation of TFE is more competitive than Gdm⁺ in either GdmSCN/TFE or GdmCl/TFE solution. The preceding accumulation of TFE around protein surface limits the approach of Gdm⁺ and water to protein. As a result, the hydrogen bonding between Gdm⁺ and water to protein is highly forbidden and the secondary structure stability of protein is strongly enhanced. In contrast, without the presence of TFE, the protein structure is largely denatured in similarly concentrated GdmSCN or GdmCl solution. Proteins 2014; 82:944–953. © 2013 Wiley Periodicals, Inc.     

Decoding the Decoding Region: Analysis of Eukaryotic Release Factor (eRF1) Stop Codon-Binding Residues

Peptide synthesis in eukaryotes terminates when eukaryotic release factor 1 (eRF1) binds to an mRNA stop codon and occupies the ribosomal A site. Domain 1 of the eRF1 protein has been implicated in stop codon recognition in a number of experimental studies. In order to further pinpoint the residues of this protein involved in stop codon recognition, we sequenced and compared eRF1 genes from a variety of ciliated protozoan species. We then performed a series of computational analyses to evaluate the conservation, accessibility, and structural environment of each amino acid located in domain 1. With this new dataset and methodology, we were able to identify eight specific amino acid sites important for stop codon recognition and also to propose a set of cooperative paired substitutions that may underlie stop codon reassignment. Our results are more consistent with current experimental data than previously described models.Han Liang, Jonathan Y. Wong,Contributed equally to this paperReviewingEditor: Dr. Niles Lehman     

An RNA replisome as the ancestor of the ribosome

Summary The ribosome is proposed to have evolved from an earlier RNA-replisome, which synthesized RNA. Ancestral tRNA molecules originally were loaded with trinucleotide sequences and donated them to growing RNA chains. The enzymatic addition of the C-C-A trinucleotide to presentday transfer RNA molecules is a carryover from this function. The strategies of reading RNA sequences by triplet codons and of housing information genetically in special repository molecules predates the origin of protein and DNA. These latter two polymers arose together at the time when the RNA replisome was converted to a ribosome.     

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine

Groups of CBA mice were administered [³⁵S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1⁰ histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HC1) contained [³⁵S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.