Translation Of Natural mRNAs Research Articles

Activities of the peptidyl transferase center of ribosomes lacking protein L27.

The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.

Rate of Translation of Natural mRNAs in an Optimizedin VitroSystem

We report results onin vitrotranslation of an mRNA coding for elongation factor TuB which wasin vitrotranscribed from the tufB gene fromEscherichia coli.Translation occurs at a rate of about 10 codons per second, which is close to thein vivorate. Protein elongation obeys Michaelis–Menten kinetics with respect to the concentrations of the elongation factors EF-Tu and EF-G in the translation system. The measuredKmvalues for EF-Tu and EF-G are 10 and 0.25 μM, respectively. The obtainedkcatandKmvalues were used to estimate the averagekcat/Kmof about 24 × 106s−1M−1for the interaction of individual EF-Tu*GTP*aa–tRNA complexes with ribosomes. The estimatedkcat/Kmvalue for EF-G is 36 × 106s−1M−1. We have also studied translation with a “hyperaccurate” ribosome variant that is pseudodependent on streptomycin (SmP). We have found that SmP ribosomes translate the TuB mRNA significantly slower than wild-type ribosomes do. This is mainly due to a threefold lowerkcat/Kmfor the interaction of EF-Tu*GTP*aa–tRNA complexes with SmP ribosomes.

Characterization of cloned measles virus mRNAs by in vitro transcription, translation, and immunoprecipitation.

A cDNA library was constructed from poly(A)+ RNA prepared from VERO cells infected with the Edmonston strain of measles virus. Clones corresponding to five major viral-specific transcripts were identified by northern blot hybridization analysis. Probes prepared from these five clones detected an additional five minor viral RNA transcripts. The sizes and hydridization patterns of these minor RNA species are consistent with their being bicistronic transcripts arising from a viral genomic template with the gene order 3'..NP-9/C-M-F-HA-(L)..5'. To assess the coding capacity of these cDNA clones they were inserted into pSP64, transcribed in vitro, the RNA was translated in reticulocyte lysates, and the protein was immunoprecipitated with specific antisera. From this analysis the genes for NP, P/C, M, F, and HA were identified. In vitro translation of natural mRNAs and SP6 transcripts of cDNAs consistently produced smaller polypeptides that appear to be initiated at internal AUGs. The relative abundance of these various cell-free translation products reflects the probability of translational initiation at the various in-frame AUGs. The patterns observed suggest that other factors besides sequences immediately flanking the AUGs have a significant effect on the selection of translational initiation sites. An increase in translational efficiency of the F transcript was achieved by removing 450 bases of the G-C-rich 5' noncoding region.

Protein synthesis in Bacillus subtilis: II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature. The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems. A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.

The purification and properties of two low-molecular-weight proteins required for the initiation of translation in ascites tumour cells.

A fractionated cell-free protein-synthesising system from Krebs II ascites tumour cells is described. It is dependent on the addition of exogenous mRNA. In this system the translation of natural mRNAs requires the presence of four initiation factors: the high-molecular-weight complex IF-M3, IFEMC and two low-molecular-weight proteins IF-Malpha and IF-Mbeta. None of these factors are required for the translation of the synthetic messenger poly(A,U,G). The purification of IF-Malpha and IF-Mbeta was achieved by the use of gel and ion-exchange chromatography. Analysis of the purified proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that the molecular weight of IF-Malpha is 19,000 and that of IF-Mbeta is 15,000. The initiation factor IF-E1 isolated from rabbit reticulocytes can substitute for IF-Mbeta in the translation of encephalomyocarditis virus RNA. IF-Malpha does not correspond to any previously recognised eukaryotic initiation factor.

Translation of natural mRNAs in vitro by extracts of a mutant in protein synthesis.

NATURAL mRNAs contain specific signals for initiation and termination of protein synthesis1,2. To further understand these processes and their regulation in the cell, we have been screening mutants defective in translation of natural mRNAs but not of artificial templates.

In vitroTranslation of Natural mRNAs in a Cell-free System Containing Components from Interferon-treated Chicken Fibroblasts and Factor Preparations from Mouse Ascites Cells or Rabbit Reticulocytes

The effect of interferon has been studied in a mixed cell-free protein synthesizing system. Hemoglobin (Hb) and Encephalomyocarditis virus (EMC)-RNA can be efficiently translated in vitro in a system containing S-30 lysates or run-off ribosomes from primary chick embryo fibroblasts (CEF) and a postmicrosomal supernatant from mouse ascites cells or a ribosomal-wash preparation from rabbit reticulocytes. Ribosomes prepared from CEF pretreated with high doses of homologous interferon (500 units/ml) were able to translate Hb-RNA in the presence of heterologous factors with the same efficiency as ribosomes prepared from control cells. Translation of EMC-RNAP WAS SLIGHTLY Reduced if ribosomes from interferon-treated cells were used in the mixed cell-free system, confirming previous reports. No inhibitory effect caused by interferon treatment of CEF cells could be detected on in vitro translation of natural mRNAs if the cells had, in addition to interferon treatment, been infected with vaccinia virus. Possible reasons for the different observations made with out cell-free protein synthesizing system from CEF and with cell-free systems prepared from mouse cells are discussed.

Interferon treatment inhibits Mengo RNA and haemoglobin mRNA translation in cell-free extracts of L cells.

THE effects of interferon on gene expression are still in debate. To explain the block in the multiplication of both DNA and RNA viruses, it was proposed that interferon affects the translation of viral mRNA, but not that of cellular or synthetic mRNAs1–5. Because active cell-free systems which translate purified mRNA were not available at the time, evidence for these effects remained suggestive but inconclusive. Very recently, marked inhibitions of virion associated transcriptases in interferon-treated cells have been reported6,7; but again, the proposed effects of interferon treatment on transcription6–8 have not been demonstrated in cell-free systems. As a cell-free system from interferon-sensitive mouse L cells, active for the in vitro translation of natural mRNAs, is now available9–11 we have reinvestigated the influence of interferon treatment on the translation of viral and cellular mRNAs.

Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.

Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.