Polypeptide Chain Elongation Research Articles

EFFECTS OF TEMPERATURE EXTREMES ON PROTEIN SYNTHESIS IN LIVER OF TOADFISH,OPSANUS TAU, IN VIVO

Amino acid incorporation and polypeptide chain elongation rates were determined in toadfish at the upper and lower ends of their range of temperature tolerance. The method was based on pulse injection of radioactive amino acids into the hepatic portal vein and analysis of ribosome-bound and completed chains at various times after injection. Elongation rates at low temperatures were obtained from the rate of completion and release of polypeptide chains pre-labeled at 20°C. Average polypeptide chain assembly time at 4°C was 6 h; that at 37°C was 1 min. Comparison with rates of total protein synthesis obtained in previous studies indicate a coordinated slowing of all protein synthetic reactions at low temperatures. A 10-fold decline in elongation rate from 7°C to 4°C suggests a specific temperature sensitivity in elongation factors or in formation of aminoacyl-tRNA. At high temperatures (32°-40°C) protein synthetic reactions show a loss of coordination, with elongation rate increasing normally (Q10 about 2) ...

Growth-rate-dependent adjustment of ribosome function in chemostat-grown cells of the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus was grown as a yeast in a chemostat. Cellular growth rates were varied over a fourfold range under an atmosphere of N2 and over an eightfold range under CO2. Under either atmosphere, an increase in the cellular growth rate resulted in increases in (i) the cellular ribosome concentration, (ii) the percentage of ribosomes active in protein synthesis, and (iii) the rate of polypeptide chain elongation. The rate of protein synthesis in this organism can therefore be regulated by adjustment of all of these mechanisms.

Further studies on the inhibition of cellular protein synthesis by vesicular stomatitis virus

After infection of L cells by vesicular stomatitis virus, the rate of protein synthesis steadily declines. Simultaneously, polysomes disaggregate and 80 S ribosomes accumulate. Transit time studies indicate that the average rate of polypeptide chain elongation is virtually identical for infected and uninfected cells. Cellular mRNAs remain intact and potentially functional in infected cells, as determined by their ability to be translated into cellular proteins in nuclease-treated reticulocyte lysates. More template activity is present in infected cells than in uninfected cells as a result of the presence of viral mRNAs in infected cells. Extracts from infected L cells translate their endogenous mRNAs less efficiently than do comparable extracts from uninfected cells. Nuclease-treated extracts from infected cells also show a lesser ability to translate given amounts of exogenous L cell or vesicular stomatitis virus mRNAs than do comparable extracts from uninfected cells. This observation suggests that the translation of both cellular and viral mRNAs is affected equally by the lesion induced in the cell by virus infection. The lesion appears to act at the level of initiation of protein synthesis and results in an underutilization of the total template activity present in infected cells.

Primary structure of the polypeptide chain elongation factor Tu from E. coli. I. Amino acid sequence of fragment B.

The complete amino acid sequence of Fragment B obtained by the limited tryptic digestion of E. coli polypeptide chain elongation factor Tu (EF-Tu) was determined. Seven peptides formed from Fragment B by cleavage with cyanogen bromide (designated as CB1 to CB7 according to their order of alignment from N- to C-termini of Fragment B) were purified, and six of them were completely sequenced by the manual method of sequential Edman degradation with direct identification of the phenylthiohydantoin-amino acids. The remaining one cyanogen bromide peptide (CB6) containing 109 amino acid residues was further digested with trypsin. Twelve tryptic peptides (designated as T1 to T12 according to their order of alignment from N- to C-termini of CB6) were isolated, and their amino acid sequences were analyzed. The alignment of CB peptides was based on the results of the automated sequence analysis of Fragment B from its N-terminal, and the sequence analysis of the overlapping peptides containing sulfhydryl groups obtained by the complete tryptic digestion of Fragment B. The alignment of peptides T1 to T12 on CB6 was based on the result of the automated sequence analysis of CB6, and the sequence of the overlapping peptide obtained by the chemical cleavage of CB6 at the tryptophan residue using cyanogen bromide in heptafluorobutyric acid. The nucleotide sequence of the tuf A gene was also utilized for the alignment of these peptides. Fragment B comprises amino acid residues 59 to 263 of E. coli EF-Tu, which consists of 393 amino acids. It contains two functional (SH1 and SH2) and one non-functional (SH3) sulfhydryl groups of EF-Tu. All of the five histidine residues in Fragment B were distributed within the first N-terminal quarter, and three of them were found to be clustered around SH2. Although E. coli EF-Tu consists of two gene products (tuf A and tuf B), no microheterogeneity was found in the amino acid sequence of Fragment B.

Studies on the high molecular weight form of polypeptide chain elongation factor-1 from pig liver. II. Interaction with guanine nucleotides and aminoacyl-tRNA.

The high molecular weight form of polypeptide chain elongation factor-1, or EF-1H, has been purified from pig liver to apparent homogeneity and shown to be a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, namely, EF-1 alpha beta gamma (Hattori, S. and Iwasaki, K. (1980) J. Biochem. 88, 725-736). Therefore, its enzymatic properties were investigated in detail. The effects of various components such as NH4Cl, Mg(CH3COO)2, guanine nucleotides and Phe-tRNA on its heat stability were investigated and it was found that its properties were very close to those of the aminoacyl-tRNA binding enzyme from rabbit reticulocytes (McKeehan, W.L. and Hardesty, B. (1969) J. Biol. Chem. 244, 4330-4339). Furthermore, it was reported that EF-1 alpha beta gamma seemed to dissociate into EF-1 alpha and EF-1 beta gamma when guanine nucleotides were present, and also to form a complex of EF-1 alpha . GTP . [14C]Phe-tRNA when both GTP and [14C]Phe-tRNA were present. On the contrary, however, when the interaction between EF-1 alpha beta gamma and guanine nucleotides was analyzed by gel filtration at 4 degrees C, the dissociation of EF-1 alpha beta gamma could not be detected, instead the formation of binary complexes containing EF-1 alpha beta gamma and guanine nucleotides was observed. The dissociation constants of the EF-1 alpha beta gamma . GDP and EF-1 alpha beta gamma . GTP complexes were estimated to be 6.8 x 10(-6) M and 7.3 x 10(-6) M, respectively, and the number of binding sites per molecule of EF-1 alpha beta gamma for these nucleotides was estimated to be one. Little, if any, interaction was detected between EF-1 alpha beta gamma and aminoacyl-tRNA even in the presence of GTP. The controversial results obtained by different methods as described above seemed to be compatible with each other, if one assumes that the dissociation constant for the conversion of EF-1 alpha beta gamma into EF-1 alpha and EF-1 beta gamma increased with the increase in temperature. When gel filtration of EF-1 alpha beta gamma was carried out with a solution containing both GTP and [14C]Phe-tRNA, the formation of a ternary complex containing EF-1 alpha, GTP and [14C]Phe-tRNA was detected at 4 degrees C, although the amount was very small. From these results, we assumed that EF-1 alpha beta gamma interacts with GTP and aminoacyl-tRNA (aa-tRNA) according to the following sequences; EF-1 alpha beta gamma + GTP in equilibrium with EF-1 alpha beta gamma . GTP EF-1 alpha beta gamma . GTP in equilibrium with EF-1 alpha . GTP + EF-1 beta gamma EF-1 alpha . GTP + aa-tRNA in equilibrium with EF-1 alpha . GTP . aa-tRNA.

The inhibition of brain protein synthesis following leucine or valine injection can be prevented

An intraperitoneal injection of either leucine (1.57 mg/g body wt) or valine (2 mg/g body wt) into newborn mice led to a rapid accumulation of inactive monoribosomes in their brains. In vitro measurements of protein synthesis by the remaining active ribosomes in leucine-treated mice revealed that polypeptide chain elongation was also inhibited. When a mixture of the seven amino acids from the leucine transport system was injected (0.15 mg each amino acid/g body wt) following the valine or leucine treatment, brain monoribosomes did not accumulate and elongation rates in the leucine-treated mice were only slightly altered.

Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes.

The previously observed (Walter, et al. 1981 J. Cell Biol. 91:545-550) inhibitory effect of SRP selectively on the cell-free translation of mRNA for secretory protein (preprolactin) was shown here to be caused by a signal sequence-induced and site-specific arrest in polypeptide chain elongation. The Mr of the SRP-arrested nascent preprolactin chain was estimated to be 8,000 corresponding to approximately 70 amino acid residues. Because the signal sequence of preprolactin comprises 30 residues and because approximately 40 residues of the nascent chain are buried (protected from protease) in the large ribosomal subunit, we conclude that it is the interaction of SRP with the amino-terminal signal peptide of the nascent chain (emerged from the large ribosomal subunit) that modulates translation and thereby causes an arrest in chain elongation. This arrest is released upon SRP-mediated binding of the elongation-arrested ribosomes to the microsomal membrane, resulting in chain completion and translocation into the microsomal vesicle.

Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos ( S. purpuratus)

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, dP∗ dt = M·f·n· A t , where dP∗ dt = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and A t = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis ( dP∗ dt ). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo −1 hr −1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo −1 hr −1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes ( f zygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes ( n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on f zygotic and dP∗ dt , the rate of elongation of nascent histone polypeptide chains ( A t ) during the first 6 hr of development was estimated; A t remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.

Selective photooxidation of histidine residues in polypeptide chain elongation factor Tu from E. coli.

When EF-Tu was photooxidized for 20 min at 0 degrees C in the presence of 10 microM GDP and 5 microM rose bengal, the activity to promote the binding of [14C]Phe-tRNA to ribosomes was rapidly lost, while the activity to bind [3H]GDP remained intact. The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of [14C]Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions. It was found, however, that these activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site(s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA . EF-Tu . GTP complex. Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu . GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged. On the other hand, no histidine residue was lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP. The photooxidized EF-Tu . GDP was then partially degraded with trypsin and each of the resulting tryptic fragments, D, B, and C (Arai, Nakamura, Arai, Kawakita, and Kaziro (1976) J. Biochem. 79, 69-83), was analyzed for histidine content. The results indicated that fragments B, C, and D had lost 0.7, 0.5, and 0.2 residues of histidine, respectively. Since fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, the above results suggest that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes.

Regulation of protein synthesis: translational control by procollagen-derived fragments.

Previous studies have shown that a type I procollagen-derived peptide, called pN alpha 1(I)-Col 1, selectively inhibits collagen synthesis by fibroblasts in culture and the translation of procollagen mRNA in a rabbit reticulocyte lysate system. We prepared the 10,700-dalton peptide from dermatosparactic calf skin, which contained high levels of incompletely processed type I procollagen, by collagenase digestion. Time-course and dose-response studies showed that the peptide specifically inhibited the translation of procollagen mRNA in preparations of human fibroblast RNA while not affecting the translation of globin mRNA or of other messenger RNAs in fibroblast RNA. After reduction and alkylation, the peptide lost its specificity but became a nonspecific inhibitor of translation. Enzymatic cleavage enabled us to localize the nonspecific activity to a short sequence -Pro-Thr-Asp-Glu, an assignment confirmed by peptide synthesis. Using pactamycin, a specific inhibitor of translational initiation, we showed that the intact peptide acts on procollagen mRNA translation by inhibition of polypeptide chain elongation or termination, or both, whereas the nonspecific inhibitory activity of the unfolded peptide and its derivatives can be attributed to an inhibition of chain initiation. Although the native peptide may function in feedback regulation of collagen synthesis, the physiological role of the lower molecular weight fragments, if any, is uncertain.

The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu.

It was previously shown that, in cell-free systems, virginiamycin M blocks the elongation of polypeptide chains by inhibiting both the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu (EF-Tu) and the peptidyl transferase reaction. The present work aims at identifying the reaction primarily affected by this antibiotic. Ribosomes, whether treated or not with virginiamycin M, are able to activate the EF-Tu GTPase under conditions in which the reaction takes place uncoupled from the binding of aminoacyl-tRNA to ribosomes. Since this antibiotic has no inhibitory action on the elongation factor G (EF-G) GTPase either, it can be concluded that the ribosomal centers for elongation factor GTPases are conclusively not affected by virginiamycin M. In the presence of kirromycin, an antibiotic that prevents dissociation of EF-Tu from the ribosome after GTP hydrolysis, virginiamycin M does not inhibit the enzymatic binding of Phe-tRNA to the poly(U)-ribosome complex or does it affect the concomitant stoichiometric hydrolysis of GTP. Under these conditions, ribosomal complexes carrying Phe-tRNA, kirromycin, EF-Tu and GDP at the A site are formed as well in the presence as in the absence of virginiamycin M. Moreover, the release of EF-Tu from ribosomes incubated with virginiamycin M was found to occur normally in the absence of kirromycin, thus suggesting an interference by the former inhibitor with an elongation step that follows the detachment of EF-Tu - GDP from the ribosomes. Indeed, aminoacyl-tRNA becomes sensitive to virginiamycin M after the release of EF-Tu, as the addition of the inhibitor at this stage promotes the release of Phe-tRNA bound enzymatically to the A site. Consequently, the accumulation of ribosomal complexes carrying an aminoacyl-tRNA at the A site is prevented, and this mimics an inhibition of the binding reaction. On the other hand, such a dissociation induced by virginiamycin M is expected to regenerate ribosomes with an empty A site, and to allow an additional round of the binding process on the same ribosome in the absence of translocation. Accordingly, the GTP hydrolysis coupled with the EF-Tu-directed binding of Phe-tRNA to the poly(U) · ribosome complex was found markedly stimulated by virginiamycin M. These observations can be accounted for by postulating that virginiamycin M acts primarily on the peptidyl transferase: the release of aminoacyl-tRNA from the A site is, then, due to an inability of the CCA-aminoacyl end of tRNA to bind to the peptidyl transferase center inactivated by the antibiotic.

Translational elongation rate changes in encephalomyocarditis virus-infected and interferon-treated cells.

Infection of mouse L cells with encephalomyocarditis virus results in a rapid inhibition of host protein synthesis before the synthesis of viral proteins. Although no alterations in initiation factor activities have been demonstrated in encephalomyocarditis virus-infected mouse cells, a defect in polypeptide chain elongation has been shown to occur in infected cell extracts. We investigated the significance of this elongation defect in the host shutoff phenomenon in vivo. Average polypeptide chain elongation rates were measured at various times after infection. Interferon was used as a reagent to separate temporarily the virus-induced alterations. Encephalomyocarditis virus infection of L cells was shown to lead to a progressive reduction in the elongation rate. Whereas interferon pretreatment delayed the decrease in elongation rate in a dose-dependent manner, it failed to alter the kinetics of host shutoff, suggesting that slowing of elongation steps played no significant role in this phenomenon. In addition, interferon pretreatment of either mock-infected or virus-infected cells led to no elongation defect that could be attributed to interferon action.

Polypeptide synthesis catalysed by components of Pichinde virus disrupted by detergent.

Pichinde virus preparations were investigated for ribosomal components and associated activities. After detergent treatment and fractionation, viral components were assayed for polypeptide synthesis, elongation of nascent polypeptide chains, and mRNA activity. It was demonstrated that these subviral particles could synthesize polypeptides when exogenous mRNA template, aminoacyl--tRNAs, translation factors, GTP and appropriate cations were added. Undisrupted, whole virions could not synthesize polypeptide; disruption and subsequent centrifugation of subviral ribosomes was a prerequisite for the biosynthetic activity described.

Rate of transacylation between 2' and 3'-O-L-phenylalanyladenosine.

The 270-MHz proton NMR spectra of 2'-O-L-phenylalanyladenosine and 3'-O-L-phenylalanyladenosine in deuterated phosphate buffer were analyzed. The transacylation between these two isomers was studied by saturation transfer experiments on H1' proton resonances and the transacylation rate was directly determined to be 0.76 s-1 at pD 6.9 and 25 degrees C. This transacylation rate is appreciably slower than the rate of polypeptide chain elongation, suggesting the presence of enzymatic activity for the transacylation of aminoacyl-tRNA in protein biosynthesis.

Regulation of protein synthesis in HEp-2 cells and their cytoplasmic extracts after poliovirus infection

In intact HEp-2 cells, the overall rate of protein synthesis decreased rapidly after poliovirus infection. Under the controlled energetic and ionic in vitro assay conditions, however, cytoplasmic extracts prepared late in infection exhibited an increased translational activity. This resulted from multiple reinitiation cycles of poliovirus RNA translation. Increased ion concentrations in the in vitro translation reaction mixtures inhibited both the rate of peptide chain initiation and elongation for cellular mRNA translation. However, excess salts inhibited the rate of polypeptide chain elongation for poliovirus in vitro translation to a higher extent than the rate of initiation. A virus-induced shut off of host cell protein synthesis was observed in vivo as well as in the in vitro translation system. Translation of cellular mRNAs in cytoplasmic extracts prepared late in infection was restored by addition of uninfected cell extract free of endogenous mRNA. We concluded that increased concentrations of ions in poliovirus-infected cells account for the decrease in the overall rate of protein synthesis, but are not involved in the mechanism of poliovirus-induced shut off of host cell protein synthesis.

Factor requirements for the tritin inactivation of animal cell ribosomes

Wheat germ contains a protein (tritin) that efficiently inhibits protein synthesis in cell-free extracts from animal cells but not from wheat germ. Tritin has been purified to apparent homogeneity and shown to block enzymatically polypeptide chain elongation. We have extended these studies to examine more closely the mechanism of tritin inactivation of animal cell ribosomes. Here we provide evidence suggesting that ATP and tRNA, previously thought to be tritin co-factors, function in the inhibition by altering the conformation of the ribosome to a form susceptible to tritin attack. Tritin treatment does not inhibit the binding of aminoacyl-tRNA to ribosomes, but it does partially reduce the ribosome binding of elongation factor 2. Tritin inhibition appears to affect the core ribosome and not ribosome-associated factors. However, the core ribosome itself is not a suitable substrate for tritin attack; a factor(s) is removed from ribosomes by high salt washing which is required for the tritin-induced inhibition. The ability to be inhibited is restored when ascites cell core ribosomes are supplemented with factors from either ascites cells or wheat germ. In contrast, neither ascites cell factors nor wheat germ factors will promote a significant tritin-induced inhibition of core ribosomes from wheat germ. This indicates that the specificity of tritin inhibition resides primarily at the level of the eukaryotic core ribosome.

Growth-rate-dependent adjustment of ribosome function in the fungus Mucor racemosus

The dimorphic fungus Mucor racemosus was grown at rates between 0.043 and 0.434 doubling/h while maintained as yeasts or at rates between 0.21 and 0.50 doubling/h while maintained as hyphae by altering the composition of the growth medium or the gaseous environment of the cells. Yeasts at the higher growth rates contained many more ribosomes than did yeasts at the lower growth rates. They also had a higher percentage of ribosomes active in protein synthesis and a faster rate of polypeptide-chain elongation than did the slower-growing cells. Hyphal cells at faster growth rates also contained many more ribosomes and showed a faster rate of polypeptide-chain elongation than did slower-growing cells. However, the faster-growing cells had a substantially lower proportion of ribosomes active in protein synthesis than did the slower-growing hyphae. Pulse-chase experiments failed to provide any evidence of protein turnover, which might otherwise invalidate the values calculated for the peptide-chain elongation rates.

The effect of inhibitors of protein and RNA synthesis on 1 alpha,25-dihydroxyvitamin D3-dependent calcium uptake in cultured embryonic chick duodenum.

To determine whether 1 alpha, 25-dihydroxyvitamin D3-dependent increases in intestinal calcium uptake require de novo protein and RNA synthesis, the effects of several inhibitors of these processes have been re-examined in vitro using cultured embryonic chick duodenum. To minimize the contributions of antibiotic toxicity to the interpretation of results, care was taken to examine inhibitor effects at early times after the onset of the 1 alpha, 25-dihydroxyvitamin D3 response. Cycloheximide at a concentration of 5 microM blocked hormone-dependent calcium uptake at all times examined (6 to 24 h). Actinomycin D was similarly effective at 6 to 12 h. The effects of cycloheximide were totally reversible while actinomycin D inhibition was only partially reversible. These compounds inhibited protein or RNA synthesis by 68.4 +/- 1.4 and 51.4 +/- 1.1%, respectively. Anisomycin, another inhibitor of polypeptide chain elongation and alpha-amanitin, an inhibitor of RNA polymerase I, also blocked 1 alpha, 25-dihydroxyvitamin D3-dependent calcium uptake after 12 h in culture. These results further strengthen the hypothesis that 1 alpha, 25-dihydroxyvitamin D3 stimulates intestinal calcium transport via a nuclear mechanism involving new gene expression.

Effect of alpha-methylphenylalanine and phenylalanine on brain polyribosomes and protein synthesis.

A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor alpha-methyl-D,L-phenylalanine (0.43 mg/g body wt). The presence of alpha-methylphenylalanine in newborn mice inhibited 65-70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40-fold in both plasma and brain following injection of alpha-methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of alpha-methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.

Comparison of the activities of HSV-1 and cellular mRNAs as templates for in vitro translation

Messenger RNA preparations from HSV-1-infected cells directed the incorporation of [ 35S]methionine in cell-free translation systems to an extent only 20–25% of that directed by uninfected cell RNA. HSV-1 mRNAs were faithfully translated and the low template activity of the RNA from infected cells could not be explained by the presence of an inhibitor. Measurements of mean ribosome transit times indicated that a reduced rate of polypeptide chain elongation on RNA from infected cells was not responsible for the poor template activity. Initiation of polypeptide chain synthesis on HSV-1 mRNAs on the other hand appeared to be less efficient than initiation on cellular mRNAs, on the basis of in vitro competition experiments. The possibility that HSV-1 mRNAs might be poor templates for protein synthesis is contrary to the situation with a number of other viruses which have been shown to have mRNAs which are particularly efficient relative to cellular mRNAs.