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H Weissbach

Publications and source records attributed to H Weissbach.

At least 127 records · Page 7Linked to original sources
PubMed1979

DNA-directed in vitro synthesis of beta-galactosidase. Purification and characterization of stimulatory factors in an ascites extract.

Previous studies have described a partially defined system for the DNA-directed in vitro synthesis of beta-galactosidase (Kung, H.F., Redfield, B., Treadwell, B.V., Eskin, B., Spears, C., and Weissbach, H. (1977) J. Biol. Chem. 252, 6889-6894). An Ehrlich ascites extract was shown in these in vitro studies to acylate Escherichia coli tRNA with 13 amino acids, and the ascites extract was used in place of the corresponding 13 E. coli aminoacyl-tRNA synthetases. The present studies indicate that the ascites extract is supplying an additional protein factor, besides the aminoacyl-tRNA synthetases, that stimulates the DNA-directed synthesis of beta-galactosidase. The protein factor has been highly purified and may be functioning by protecting mRNA against degradation. In addition, NAD or T4 DNA ligase stimulates the synthesis of beta-galactosidase in the partially defined system.

Amino Acyl-tRNA Synthetases↗
PubMed1979

DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.

Bacterial Proteins↗
PubMed1979

In vitro regulation of DNA-dependent synthesis of Escherichia coli ribosomal protein L12.

The DNA of the transducing phage lambdarifd18 contains, among others, the genes for the ribosomal proteins L11, L1, L10, and L12 and the beta and beta' subunits of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). In a coupled in vitro protein-synthesis system, lambdarifd18 DNA directs the synthesis of about four to five molecules of L12 per molecule of L10. This is consistent with the finding that there are four copies of L12 per ribosome. The ratio of L12/L10 was also examined from an EcoRI fragment of lambdarifd18 that contains the L10 gene and about 50% of the L12 gene. A significantly lower ratio of truncated L12/L10 was observed compared to the intact phage. The binding of RNA polymerase to various lambdarifd18 DNA restriction fragments was used to locate possible promoter sites. These binding experiments suggest that the beta and beta' subunits of RNA polymerase are cotranscribed with at least ribosomal protein L12 and, also, that there may be an additional promoter site for the L12 gene within the structural gene for L10.

Coliphages↗
PubMed1979

Reduction of methionine sulfoxide to methionine by Escherichia coli.

L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine. This was verified by showing that a cell-free extract of E. coli catalyzes the conversion of methionine sulfoxide to methionine. This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound. The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml.

Escherichia coli↗
PubMed1978

Binding of reticulocyte elongation factor 1 to ribosomes and nucleic acids.

The present study has examined the requirements for the binding of rabbit reticulocyte elongation factor 1 (EF-1) to ribosomes under different assay conditions. When a centrifugation procedure was used to separate the ribosome EF-1 complex, the binding of EF-1 to ribosomes required GTP and Phe-tRNA, but not poly(U). The results suggested that undr these conditions a ternary complex, EF-1 . GTP . aminoacyl-tRNA, is necessary for the formation of a ribosome . EF-1 complex. However, when gel filtration was used to isolate the ribosome . EF-1 complex, only template and tRNA were required. These studie emphasize the fact that the procedure used to isolate the ribosome . EF-1 complex determines the requirements for stable complex formation. EF-1 can also interact with nucleic acids such as 28 S and 18 S rRNA, messenger RNA and DNA. In contrast to the binding to ribosomes, EF-1 binding to nucleic acids requires only Mg2+.

Animals↗
PubMed1978

Modification of rat liver RNA polymerase I after in vivo stimulation by hydrocortisone or methylisobutylxanthine.

Following in vivo administration of hydrocortisone or methylisobutylxanthine to rats, higher levels (1.5- to 2.3-fold) of RNA polymerase I activity are present in liver nuclei and nucleoli of the treated animals as compared to control animals. The elevated specific activity is retained after purification of the enzyme under conditions where the enzyme is dependent on exogenous template for activity. The elevated polymerase activity in nuclei, nucleoli, and soluble enzyme can be destroyed by mild trypsin treatment which results in a rapid decay of the specific activity to the control level. Under these conditions, the control polymerase I activity is stable. The results indicate that in vivo stimulation by hydrocortisone or methylisobutylxanthine results in a conversion of the enzyme to a form that is catalytically more active but has an increased sensitivity to proteolysis.

Animals↗
PubMed1978

DNA-directed in vitro synthesis of Escherichia coli beta-isopropylmalate dehydrogenase.

The in vitro synthesis of beta-isopropylmalate dehydrogenase (EC 1.1.1.85), an enzyme involved in leucine biosynthesis, has been obtained using as template DNA from the hybrid plasmid (pLC1) which contains the Escherichia coli leucine operon. Enzyme synthesis in vitro is stimulated about 2-fold by guanosine-5'-diphosphate-3'-diphosphate and inhibited about 60% by 2 X 10(-4) M L-leucine.

Alcohol Oxidoreductases↗
PubMed1978

Oxidation of the methionine residues of Escherichia coli ribosomal protein L12 decreases the protein's biological activity.

Oxidation of ribosomal protein L12 with hydrogen peroxide converts the three methionine residues to methionine sulfoxide. The oxidized protein has a decreased ability to bind to ribosomes, interact with ribosomal protein L10, be precipitated by L12 antiserum, and serve as substrate for the acetylating enzyme that converts L12 to L7. Full activity of L12 is regained when the protein is reduced with 2-mercaptoethanol. Sedimentation equilibrium analysis shows that oxidation of the methionine residues in L12 causes the conversion of the protein from the dimer to the monomer form, and the results indicate that the dimer is the active form of the protein in the above reactions.

Alkylation↗
PubMed1977

DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors.

The phage DNA-directed synthesis of beta-galactosidase has been examined in a system containing the following purified Escherichia coli factors: RNA polymerase; cyclic AMP receptor protein; N10-formyltetrahydrofolate Met-tRNAf transformylase; initiation factors 1, 2, and 3; elongation factors Tu, Ts, and G; release factors 1 and 2; 20 aminoacyl-tRNA synthetases; L factor (Kung, H. F., Spears, C., and Weissbach, H. (1974) J. Biol. Chem. 250, 1556-1562); and Lalpha (Kung, H.-F., Spears, C., and Weissbach, H. (1976) Fed. proc. 35, 1537). Under these conditions, beta-galactosidase synthesis occurs at less than 1% of the rate obtained with unfractionated extracts, which suggested that other required components were lacking. The difficulty in obtaining large amounts of the purified aminoacyl-tRNA synthetases for these studies made it necessary to modify the system. It was possible to conserve many of the purified aminoacyl-tRNA synthetases since at least 13 of them could be replaced by an Ehrlich ascites extract. The ascites extract plus other E. coli purified factors was used as a basic system to search for additional components required for beta-galactosidase synthesis. The present report describes the purification from E. coli extracts of three fractions, called Lbeta, Lgamma, and Ldelta, that are needed to restore enzyme synthesis.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Effect of ribosomal proteins on synthesis and assembly of preribosomal particles in isolated rat liver nuclei.

Ribosomal proteins are rapidly taken up by isolated rat liver nuclei. The proteins are localized mainly in the nucleolus and are found associated with a nucleolar 80 S particle containing newly synthesized 45 S RNA. Ribosomal proteins in the nucleoplasm were associated with particles containing 18 and 28 S RNAs. In the absence of ribosomal proteins in the incubations, there was a decrease in the amount of newly synthesized 45, 18, and 28 S RNAs and an increase in low molecular weight RNA in both the nucleolus and nucleoplasm. This in vitro system appears to be useful for studies on the formation of ribosomal particles in the nucleus.

Animals↗