PubMed Health⌕ Search

Biomedical subjects

H Weissbach

Publications and source records attributed to H Weissbach.

At least 91 records · Page 5Linked to original sources
PubMed1984

In vitro regulation of phage lambda cII gene expression by Escherichia coli integration host factor.

The effect of Escherichia coli integration host factor (IHF) on phage lambda gene expression has been examined in a simplified DNA-directed in vitro system that measures the formation of the first dipeptide of the gene product. Plasmid pKC30cII, which contains the phage lambda genes N, cII and O, under control of the PL promoter, was used as template to study the expression of the first dipeptide of the gene products--i.e., fMet-Asp for N protein, fMet-Val for cII, and fMet-Thr for O. Purified IHF stimulates the DNA-directed synthesis of fMet-Val (cII) and fMet-Thr (O) 2-3-fold but has no effect on the synthesis of fMet-Asp (N). In this in vitro system, the stimulation by IHF of cII and O gene expression is at the level of transcription. Phage lambda repressor completely inhibits dipeptide synthesis in the presence or absence of IHF. The results are consistent with a role of IHF as a transcription antiterminator, perhaps functioning at or near the tR1 site preceding the cII gene.

Bacteriophage lambda↗
PubMed1984

Two initiation sites detected in the small s1 species of reovirus mRNA by dipeptide synthesis in vitro.

Reovirus mRNAs directed the synthesis of fMet dipeptides in a translation initiation system reconstituted from rabbit reticulocyte initiation and elongation factors, Artemia salina 80S ribosomes, yeast fMet-tRNAiMet and Escherichia coli3H-labeled aminoacyl tRNAs. As predicted from the GC(U,G) codon that follows the 5'-proximal AUG in half of the viral mRNA species, fMet-Ala was the predominant dipeptide product obtained in response to a mixture of mRNAs or to the separated size classes of medium (m) and small (s) mRNA. The four individual small mRNA species each directed the synthesis of an fMet dipeptide that was consistent with the utilization of the 5'-proximal AUG for initiation. In addition to fMet-Asp, the s1 mRNA also directed fMet-Glu synthesis indicative of initiation in a second reading frame at the 5'-penultimate AUG. The tripeptide fMet-Glu-Tyr was also synthesized from s1 mRNA, which further verified this second initiation site. mRNAs containing 5'-terminal GpppG were 10-15% as active as the corresponding m7G-capped templates. The dipeptide assay provides a rapid method for determining initiation sites in individual mRNAs or in mixtures of mRNAs.

Amino Acid Sequence↗
PubMed1983

Translational control of the expression of the beta subunit gene of E. coli RNA polymerase.

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the in vitro synthesis of fMet-Val, the N-terminal dipeptide of the beta subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of beta, beta 1 synthesis is at the level of translation. L factor (nusA gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.

Bacterial Proteins↗
PubMed1983

A coupled DNA-directed in vitro system to study gene expression based on di- and tripeptide formation.

In this report, a simplified coupled DNA-directed in vitro system has been described that is based on the formation of the first di- or tripeptide of the gene product. This system is gene specific and quantitative, and the assay (especially the extraction procedure) is very rapid. The fact that both transcription and translation initiation occur in this system makes it ideally suited for studies on the regulation of prokaryotic gene expression. The ideal templates are plasmids, DNA fragments or purified mRNAs that direct the synthesis of a limited number of products with different second amino acids. An essential requirement is that the initial sequence of the protein products be known, although this system could be used to determine the second amino acid in cases where there is some doubt from the DNA sequence as to where a particular protein initiates. A difficulty arises when a plasmid contains more than one gene whose protein products have the same initial dipeptide. One solution to the problem is to measure tripeptide formation if the third amino acid is different. A second procedure, if the code word for the second amino acid differs between the genes, is to use purified isoacceptor tRNA species to distinguish the products. Another important application of tripeptide synthesis is that it can be used as a measure of the amount of active mRNA present in a mixture of mRNAs. The use of a ribosomal high-salt wash instead of the purified initiation and elongation factors greatly simplifies this system and should make it suitable for routine analysis in most laboratories.

DNA↗
PubMed1983

In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.

We used an in vitro DNA-dependent protein-synthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNA-dependent peptide-synthesizing system, synthesis of the di- and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site.

Bacterial Proteins↗
PubMed1983

Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.

An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNAi, and rabbit liver or Escherichia coli Val-tRNA1Val. Picomole quantities of fMet-Val are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMet-Val-His are synthesized, corresponding to the NH2-terminal sequence of alpha- and beta-globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the alpha- and beta-globin mRNA species.

Animals↗
PubMed1983

Oxidation of methionine residues in proteins of activated human neutrophils.

A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins.

Amino Acids↗
PubMed1983

Human methionine sulfoxide-peptide reductase, an enzyme capable of reactivating oxidized alpha-1-proteinase inhibitor in vitro.

The present study demonstrates the presence of methionine sulfoxide [Met(O)] peptide reductase activity in human lung homogenates and in lysates of polymorphonuclear leukocytes (PMN) and alveolar type II cells. Enzyme activity was not detected in human bronchoalveolar lavage fluid or in pulmonary alveolar macrophage lysates. The Met(O)-peptide reductase derived from PMN is capable of reactivating alpha-1-proteinase inhibitor (alpha 1Pl) oxidized by treatment with chloramine-T or a myeloperoxidase oxidizing system. However, the PMN-derived enzyme does not reactivate alpha 1Pl inactivated by treatment in vitro with aqueous solutions of cigarette smoke plus peroxide. In addition, after the instillation of oxidized human alpha 1Pl into lungs of normal or ozone-tolerant rats, no reactivated alpha 1Pl could be found in the pulmonary lavage obtained from these animals. Finally, patients with chronic obstructive pulmonary disease appear to have normal levels of PMN Met(O)-peptide reductase.

Animals↗
PubMed1982

In vitro synthesis of the first dipeptide of the beta subunit of Escherichia coli RNA polymerase.

Plasmids pNF1337 and pNF1341, which contain part of the L10 operon including the RNA polymerase beta-subunit gene, have been used as templates in vitro to investigate expression of the beta-subunit gene. For these studies, the synthesis of the first dipeptide of the beta subunit, fMet-Val, was measured instead of that of the entire protein. By using this dipeptide system, we studied the effects of RNA polymerase holoenzyme and L factor (nus A gene product) on fMET-Val synthesis and compared the relative effects of the primary and secondary promoters in the L10 operon on expression of the beta-subunit gene. The results show that the inhibitory effect of RNA polymerase on beta-subunit synthesis and the stimulatory effect of L factor occur before formation of the first dipeptide bond. In this in vitro system, the secondary promoters account for about 50% of the total fMet-Val synthesized. Although the primary promoter is sensitive to guanosine 5'-diphosphate 3'-diphosphate in vitro, the secondary promoters are not affected by this nucleotide.

Bacterial Proteins↗
PubMed1982

Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

A simplified translation system coupled to DNA transcription that involves assaying the synthesis of the first dipeptide of a gene product has been described recently [Robakis, N., Meza-Basso, L., Brot, N. & Weissbach, H. (1981) Proc. Natl. Acad. Sci. USA 78, 4261--4264]. Using this dipeptide system, we have investigated the expression of genes carried on plasmids coding for beta-lactamase, ribosomal protein L12, and the chloroplast large subunit (LS) of ribulosebisphosphate carboxylase (RbuBPCase). Although all three nascent gene products begin with the sequence fMet-Ser, the formation of fMet-Ser can be used to distinguish between the synthesis of beta-lactamase and either L12 or the LS of RbuBPCase by using different serine isoacceptor tRNA species. In beta-lactamase, the serine codon is AGU, which utilizes the serine isoacceptor species tRNASer3; in L12 and the LS of RbuBPCase, the serine codewords are UCU and UCA, respectively, both of which are recognized by the serine isoacceptor species tRNASer1. By using either pure tRNASer1 or pure tRNASer3, the expression of each gene can be quantitated. In this system, guanosine-5'-diphosphate-3'-diphosphate inhibits the expression of the beta-lactamase and L12 genes but stimulates the synthesis of the LS. In addition, the ratio of fMet-Ser/fMet-Ala (L12/L10) synthesized was about 1 as compared with the ratio of 4 that has been obtained previously in vivo or in vitro protein-synthesizing systems in which the entire gene product was measured.

Base Sequence↗
PubMed1981

Purification of Euglena gracilis chloroplast elongation factor Ts.

Previous studies have demonstrated that the activity of Euglena gracilis chloroplast elongation factor Ts is regulated by light and the gene for this factor is nuclear coded (Fox, L., Erion, J., Tarnowski, J., Spremulli, L., Brot, N., and Weissbach, H., J. Biol. Chem. (1980) 255, 6018-6019). Chloroplast elongation factor Ts has been purified to near homogeneity from E. gracilis extracts prepared from light-grown cells. It is composed of a single polypeptide chain of approximately 62,000 daltons. It interacts stoichiometrically with Escherichia coli elongation factor Tu to form an elongation factor Tu.elongation factor Ts complex.

Bacterial Proteins↗