Biomedical subjects

R E Thach

Publications and source records attributed to R E Thach.

At least 37 records · Page 2Linked to original sources

PubMed1988

Discriminatory interaction of purified eukaryotic initiation factors 4F plus 4A with the 5' ends of reovirus messenger RNAs.

The interaction of several reovirus mRNAs with cap-binding initiation factors has been investigated. Two quantitative experimental techniques have been applied to this question: (a) the rates of reaction of different mRNAs with tobacco acid pyrophosphatase and (b) the extent of cross-linking of different mRNAs to initiation factors in the presence and absence of ATP. The effects of ionic strength on these reactions have also been investigated. Our results demonstrate for the first time that the purified initiation factors interact differentially with purified reovirus mRNAs under competitive conditions and thus confirm earlier interpretations based on kinetic data. Comparison of the data from these studies with the translational behavior of the reovirus mRNAs, both in vitro and in vivo, has also led to specific predictions about features of these mRNAs that determine their competitive efficiencies. 1) Under ordinary ionic conditions, the steric accessibility of the m7G cap moiety of a reovirus mRNA appears to be a major determinant of its translation rate. 2) When the ionic strength is increased to supranormal levels, an additional feature, which may simply be the amount of secondary structure formed by sequences proximal to the cap, can become rate-limiting for several, but not all, of these mRNAs.

Animals↗

PubMed1988

Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors.

Three mammalian eukaryotic initiation factors (eIF) are required for the ATP-dependent binding of mRNA to the 40 S ribosomal subunit. These three factors, eIF-4A, eIF-4B, and eIF-4F, have also been isolated from wheat germ. Three assays were used to measure the ability of the wheat germ factors to interact with and/or substitute for the mammalian factors. Two assay systems were used to measure partial reactions involving the interaction of the three factors, ATP, and mRNA: 1) RNA-dependent ATP hydrolysis and 2) cross-linking of the factors to the 5' cap of oxidized mRNA. A third assay system was used to measure the ability of the factors to support initiation of protein synthesis. The results of the ATP hydrolysis and cross-linking experiments indicate that the wheat germ factors can interact with or substitute for the mammalian factors. Wheat germ eIF-4A appears to be functionally equivalent to mammalian eIF-4A. Wheat germ eIF-4B and eIF-4F appear to be isozymes possessing functions similar to mammalian eIF-4F. Wheat germ eIF-4B does not appear to be a functional equivalent to the mammalian eIF-4B. In a complete translation system from wheat germ, mammalian factors partially substitute for wheat germ factors, whereas the wheat germ factors are ineffective in the mammalian system.

Adenosine Triphosphate↗

PubMed1988

Selective inhibition of proteins synthesized from different mRNA species in reticulocyte lysates containing L-pyrroline-5-carboxylic acid.

L-Pyrroline-5-carboxylic acid is a naturally occurring nonprotein amino acid present in human plasma that changes concentrations with diet. L-pyrroline-5-carboxylic acid inhibited net synthesis of globin in untreated reticulocyte lysates in a dose dependent manner. This inhibition was greater than that observed with equimolar GSSG or NADP+ and was prevented by a NADPH generating system. L-pyrroline-5-carboxylic acid also inhibited net synthesis of proteins from brome mosaic and alfalfa mosaic virus mRNAs to different extents. However, no effect on the translation of the naturally uncapped encephalomyocarditis virus mRNA was observed. In general, mRNAs that are considered strongly competitive, such as alfalfa mosaic virus 2 and 4, were more resistant to this inhibitory process. These results indicate that pyrroline-5-carboxylic acid can initiate a differential effect on proteins synthesized from different mRNA species by an as yet unidentified mechanism.

Animals↗

PubMed1988

Translational repression in eukaryotes: partial purification and characterization of a repressor of ferritin mRNA translation.

Mouse and rabbit ferritin mRNAs translate very poorly in rabbit reticulocyte lysates relative to most other mRNAs. This translational deficiency is not seen in wheat germ lysates, suggesting the presence of an inhibitor in reticulocyte lysate that is specific for ferritin mRNA. A specific repressor of ferritin mRNA translation has been partially purified from rabbit reticulocytes by differential ultracentrifugation, ammonium sulfate fractionation, and chromatography on phosphocellulose, DEAE-cellulose, and Sephacryl S-300. The elution profile from the latter suggests an aggregate molecular mass of approximately 180 kDa for the repressor. The inhibitory activity of this repressor against native ferritin mRNA can be relieved by adding in vitro transcripts of ferritin light-chain RNAs that contain the first 92 nucleotides of the 5' untranslated region. No other sequences appear to be necessary for this effect.

Animals↗

PubMed1987

The ATP-dependent interaction of eukaryotic initiation factors with mRNA.

The interaction of three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F, with mRNA has been examined. Three assays specifically designed to evaluate this interaction are RNA-dependent ATP hydrolysis, retention of mRNAs on nitrocellulose filters, and cross-linking to periodate-oxidized mRNAs. The ATPase activity of eIF-4A is only activated by RNA which is lacking in secondary structure, and the minimal size of an oligonucleotide capable of effecting an optimal activation is 12-18 bases. In the presence of ATP, eIF-4A is capable of binding mRNA. Consistent with the ATPase activity, this binding shows a definite preference for single-stranded RNA. In the absence of ATP, eIF-4F is the only factor to bind capped mRNAs, and this binding, unlike that of eIF-4A, is sensitive to m7GDP inhibition. The activities of both eIF-4A and eIF-4F are stimulated by eIF-4B, which seems to have no specific independent activity in our assays. Evidence from the cross-linking studies indicates that in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F binds to the 5' cap region of the mRNA. From the data presented in conjunction with the current literature, a suggested sequence of factor binding to mRNA is: eIF-4F is the first initiation factor to bind mRNA ind an ATP-independent fashion; eIF-4B then binds to eIF-4F, if in fact it was not already bound prior to mRNA binding; and finally, eIF-4A binds to the eIF-4F X eIF-4B X mRNA complex and functions in an ATP-dependent manner to allow unwinding of the mRNA.

Adenosine Triphosphatases↗

PubMed1987

Procedures for enhancing the utility of the metallothionein promoter for the regulated expression of downstream open reading frames.

Procedures which enhance the inducibility of the mouse metallothionein I (mMT-I) transcriptional promoter in mouse C127 cells transformed by bovine papilloma virus have been investigated. These include: (i) induction with Zn2+ at low serum concentration, and (ii) use of a 'superinduction' protocol (presence of 1 microgram/ml of cycloheximide during induction with Zn2+, followed by 2 micrograms/ml of actinomycin D). Use of procedure (i) alone gave a 15- to 20-fold induction of expression of a downstream open reading frame (ORF), which is comparable to the maximum inducibility achieved with mMT-I in other systems. Use of procedures (i) and (ii) in combination allowed a 50-fold induction. Three different reporter ORFs (rabbit ferritin L subunit, human chorionic gonadotropin alpha subunit, and human lutropin beta subunit), in three different chromosomal contexts, responded to these procedures. The maximum rate of expression achieved was estimated at over 10(9) molecules per cell per day, which is 20% of the transformed cell's protein synthetic capacity. At these extremely high levels some of the induced products were cytotoxic.

Animals↗

PubMed1986

Influence of 5' proximal secondary structure on the translational efficiency of eukaryotic mRNAs and on their interaction with initiation factors.

The effects of 5' proximal secondary structure in mRNA molecules on their translation and on their interaction with the eukaryotic initiation factors (eIF)-4F, eIF-4A, and eIF-4B have been examined. Secondary structures were generated in the 5' noncoding region of rabbit globin and reovirus mRNAs by means of hybridization with cDNA molecules. cDNAs hybridized to the first 15 bases downstream from the cap inhibited the translation of the mRNAs in both reticulocyte and wheat germ lysates. The degree of inhibition was directly related to the monovalent ion concentration and inversely related to reaction temperature. These hybrid structures also reduced the competitive ability of the messages. Hybrid structures beginning downstream from the first 15 bases did not inhibit the translation of beta-globin mRNA or reovirus s3 mRNA. None of the hybrid structures were detrimental to the interaction of the mRNAs with the 26-kDa cap binding protein of eIF-4F, as determined by chemical cross-linking assays. However, in the presence of ATP, hybrid structures immediately adjacent to the cap severely inhibited the cross-linking to the p46 subunit of eIF-4F or to additional eIF-4A or eIF-4B. In order to account for these observations, a two-step mechanism is proposed for the interaction of eIF-4F with the 5' end of an mRNA molecule. The first step involves a weak initial interaction of the p26 subunit with the cap. The second step requires the hydrolysis of ATP and results in the formation of a stable initiation factor-mRNA complex, which may involve eIF-4A and eIF-4B. This second step is inhibited by the presence of 5' proximal secondary structure. In any event, our results demonstrate that the effect of mRNA structure on translation rate depends strongly on its position with respect to the 5' end and that this effect is due at least in part to an inhibition of the action of initiation factors normally required for the unwinding of structure.

DNA↗

PubMed1986

Recycling of messenger RNA cap-binding proteins mediated by eukaryotic initiation factor 4B.

The ability of polypeptide components of eukaryotic initiation factor (eIF) 4F to bind to the m7G cap of an mRNA, to be released from that mRNA, and then to rebind to the cap of a second mRNA has been investigated. The release and rebinding steps have been termed "recycling." It was found that eIF-4B stimulates the recycling of the 24-26 kDa (p24) component of eIF-4F, and perhaps of other components as well. By contrast, eIF-4A seemed to have little or no effect on the recycling of eIF-4F components, either in the presence or absence of eIF-4B. The recycled p24 is capable of cross-linking to oxidized cap structures. The recycled factor is also able to stimulate the cross-linking of added eIF-4A, which cross-links poorly in the absence of eIF-4F. By these criteria it seems likely that the recycled eIF-4F components are active for a second round of translational initiation.

Animals↗

PubMed1986

Translational control of gene expression in a normal fibroblast. Characterization of a subclass of mRNAs with unusual kinetic properties.

The translation of a small number of mRNAs in mouse SC-1 fibroblasts can be stimulated by cycloheximide, under conditions where the synthesis of most proteins is inhibited. These mRNAs are ordinarily present in small polyribosomes or messenger ribonucleoprotein particles, although the addition of cycloheximide drives them into large (greater than or equal to 5) polysomes. These mRNAs cannot be translated in vitro unless they are extracted with phenol. With such treatment, however, they are translated with normal competitive efficiencies. In iron-poor media, the mRNA for ferritin exhibits several of the distinctive kinetic properties of this class of mRNAs. With iron supplementation, however, ferritin translation appears normal. These observations are consistent with the existence of translational induction/repression systems in eukaryotes. Several types of evidence suggest that repressors may act by interfering with the interaction between mRNAs and limiting translational initiation components.

Animals↗

PubMed1985

Effect of medium hypertonicity on reovirus translation rates. An application of kinetic modeling in vivo.

Translation rates were determined for host and virus mRNAs in reovirus-infected SC-1 cells in hypertonic medium. The effect of low doses of cycloheximide on these translation rates was also measured. The results show that hypertonicity selectively stimulates viral translation relative to host translation. Moreover, in hypertonic medium, host translation is slightly stimulated by low doses of cycloheximide, whereas viral translation is markedly inhibited. This effect of cycloheximide is precisely the opposite to what was previously observed in isotonic media [Walden, W. E., Godefroy-Colburn, T., & Thach, R. E. (1981) J. Biol. Chem. 256, 11739-11746]. It is shown that both these effects of hypertonicity are predicted by the message competition/discrimination model previously described and thus provide support for the applicability of certain aspects of the model to translation rates in vivo.

Animals↗

PubMed1985

ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors.

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or "unwind" mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.

Adenosine Triphosphate↗

PubMed1985

Shutoff of host translation by encephalomyocarditis virus infection does not involve cleavage of the eucaryotic initiation factor 4F polypeptide that accompanies poliovirus infection.

Studies were conducted to determine whether encephalomyocarditis virus infection causes proteolytic cleavage of any of the polypeptides which comprise eucaryotic initiation factor 4F. Since no such alterations in the components of the initiation factor were detected, these observations confirmed that the mechanisms whereby encephalomyocarditis virus and poliovirus shut off host translation are different.

Animals↗

PubMed1983

Unusual requirements for optimum translation of polio viral RNA in vitro.

The translation of poliovirion RNA (polio RNA) in an in vitro fractionated system was much less efficient than that of encephalomyocarditis virion RNA (EMC RNA). In contrast, when polio and EMC RNAs were added to postmitochondrial cell lysates (S10), they were translated with equal efficiency. However, this equality was observed only when high concentrations of S10 were employed; at lower concentrations, polio RNA translation was reduced relative to that of EMC RNA. These results suggest that both the fractionated and S10 systems are limiting in a component that is required for the optimal translation of polio RNA. The elongation rates for EMC and polio RNA translation in the fractionated system were found to be similar, indicating that this component acts at an initiation step. Various components, including excess ribosomal salt wash and postribosomal supernatant of cell lysate, were added to the fractionated system in an effort to identify the slow step more precisely. Of these, only excess ribosomal salt wash specifically stimulated polio RNA translation, suggesting that one or more initiation factors is necessary in unusually large amounts for this mRNA. Various purified initiation factors were tested for the ability to enhance polio RNA translation. Of these, only purified eukaryotic initiation factor 4A had a specific effect. This suggests that polio RNA, in contrast to other mRNAs tested (EMC, reoviral, and globin), may have an unusually low affinity for this initiation factor. The significance of these results is discussed in terms of the methods picornaviruses have evolved for reprogramming the translational machinery of the host cell.

Animals↗

PubMed1983

Role of mRNA competition in regulating translation: further characterization of mRNA discriminatory initiation factors.

Host and reovirus mRNAs compete with one another for translation in infected cells. Kinetic analysis has suggested that the site of competition is a message discriminatory initiation factor which must bind to the mRNA before it can interact with the 40S ribosomal subunit. The present communication describes an in vitro assay which can detect message discriminatory activities. A competitive situation is established by using reovirus and globin mRNAs, and then the specificity with which this competition is relieved by added components is measured. Among the various initiation factors surveyed with this assay, two have the properties expected of the mRNA discriminatory factor. These are eukaryotic initiation factor 4A and a "cap binding protein" complex. Inasmuch as the cap binding protein complex contains a subunit similar or identical to the initiation factor eIF-4A, it seems likely that only one form of the latter factor may be active in vivo. In vitro, both factors relieve competition among both capped and uncapped reovirus mRNAs according to similar hierarchies. These results suggest that some feature other than the m7G cap, such as nucleotide sequence or secondary structure, is recognized by the discriminatory factor.

Animals↗

PubMed1982

Translational specificity in reovirus-infected mouse fibroblasts.

The specificity of protein synthesis in reovirus-infected mouse SC-1 cells was investigated, with the following results: a, extracts from infected and control cells translated saturating levels of capped globin mRNA with the same efficiency; b, infected cell extracts did not display a virus-induced increase in the capacity to translate decapped globin mRNA; c, translation of both cellular and viral messages was equally sensitive to the cap analog, 7-methylguanosine 5'-triphosphate. These findings are consistent with a previously published model (Walden, W., Godefroy-Colburn, T., and Thach, R. E. (1981) J. Biol. Chem. 256, 11739-11746) in which translation rates in reovirus-infected SC-1 cells are regulated by competition of capped host and viral mRNAs for a component of the unaltered protein synthetic system. Similar experimental results were obtained using reovirus-infected mouse L cells, with the following exceptions: a, the shutoff of host translation was far greater in L cells than in SC-1 cells; b, extracts of infected L cells were less active than controls for translation of all mRNAs tested. The reasons for the differences between SC-1 cells and L cells in their response to reovirus infection are being investigated.

Animals↗

PubMed1982

Inhibition of host translation in encephalomyocarditis virus-infected L cells: a novel mechanism.

Encephalomyocarditis virus induced a rapid shutoff of host translation in mouse L cells shortly after infection and before viral proteins were made in detectable amounts. This kinetic pattern is similar to that seen in poliovirus-infected HeLa cells. However, the mechanisms of host shutoff are different in these two cases, for no reduction in the ability of lysates from encephalomyocarditis virus-infected L cells to translate capped mRNAs was observed. Instead, a change in the subcellular distribution of one or more initiation factors was seen. In particular, cap recognition activity in the high-speed supernatant fraction (S200) prepared from cell lysates increased threefold as a result of virus infection. The significance of this observation in terms of possible shutoff mechanisms is discussed. Inasmuch as the rapid host shutoff is not induced in at least four other cell types by encephalomyocarditis virus infection, it may be concluded that host shutoff mechanisms not only vary within the picornavirus group, but also depend upon the particular cell type employed.

Animals↗

PubMed1981

The role of mRNA competition in regulating translation. III. Comparison of in vitro and in vivo results.

Competition of encephalomyocarditis virus, reovirus, and L-cell mRNAs for a message-discriminatory component was studied in vitro. The data were analyzed qualitatively to determine the relative initiation efficiencies among the various mRNAs. The effects of potassium chloride concentration, magnesium acetate concentration, and m7G methylation on mRNA competition in vitro were also studied. These results were correlated with translation rates in vivo for the same mRNAs, to determine if the sites of competition in vitro and in vivo are the same. It was found that under a particular set of magnesium acetate and potassium chloride concentrations, the order of mRNA initiation efficiencies was the same both in vivo and in vitro, suggesting that the same limiting message-discriminatory factor is regulating initiation rates in both cases. This can only be accomplished in a competitive situation when RNA is in molar excess relative to the discriminatory component.

Animals↗

PubMed1981

The role of mRNA competition in regulating translation. I. Demonstration of competition in vivo.

SC-1 cells infected with reovirus continue to synthesize host proteins at a nearly normal rate for at least 12 h postinfection. The relative translational efficiencies of reovirus and host mRNAs were determined at various times during this interval. Analysis was performed by determining the sensitivity of individual mRNA translation rates to inhibition by low doses of cycloheximide, by measuring polysome sizes, and by quantitatively comparing mRNA concentration with protein synthesis rates. Treatment of infected cells with low doses of cycloheximide actually stimulates the synthesis of some of the major reovirus protein, whereas host protein synthesis is inhibited. An analysis of the polysomes encoding two reovirus proteins (microNS and sigma 3; synthesis of both is stimulated by cycloheximide) shows that these proteins are synthesized on polysomes smaller than those producing host proteins of comparable size. Reovirus mRNAs accumulate to unusually high levels during the first 8 h of infection, accounting for 30% to 45% by weight of the total mRNA in the cell. We interpret these results as indicating that viral and host mRNAs must compete for a message-discriminatory component prior to their binding to the 40 S ribosomal complex and that this component is limiting in reovirus-infected cells. Moreover, a hierarchy must exist among mRNAs in terms of their affinity for this component, host mRNAs in general having greater affinities than reovirus mRNAs.

Animals↗