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Biomedical subjects

D Valenzuela

Publications and source records attributed to D Valenzuela.

25 records · Page 2Linked to original sources
PubMed1981

Reversible inactivation of Escherichia coli methionyl-tRNA synthetase by covalent attachment of formylmethionine tRNA to the tRNA binding site with a cleavable cross-linker.

Protein affinity labeling groups have been attached to single-stranded cytidine residues in four structural regions of tRNAfMet. Modification of the tRNA with an average of one cross-linking group per molecule is achieved with retention of 75% of the original methionine acceptor activity. Incubation of the modified tRNA with methionyl-tRNA synthetase (MetRS) results in covalent coupling of the protein and nucleic acid by reaction of N-hydroxysuccinimide ester groups attached to the tRNA with lysine residues in the enzyme. In the presence of excess MetRS, approximately 30% of the input tRNA can be covalently bound to protein, indicating that lysine residues are appropriately oriented for reaction with cross-linking groups attached to certain sites in the tRNA but not to others. The cross-linking reaction results in loss of aminoacylation activity of MetRS equal to the amount of covalently bound tRNA. Enzyme activity is restored by release of bound tRNA following cleavage of the disulfide bond of the cross-linker with a sulfhydryl reagent. The data indicate that cross-linking occurs at the tRNA binding site of the enzyme. In the presence of excess modified tRNAfMet, a maximum of 1 mol of tRNA is cross-linked per mol of MetRS, in keeping with the known anticooperative tRNA binding properties of the native dimeric synthetase. In addition, the coupling reaction is effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAs.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Rabbit reticulocyte initiation factor 2 contains two polypeptide chains of molecular weights 48,000 and 38,000.

Eukaryotic initiation factor 2 (eIF-20) purified from rabbit reticulocyte lysates consists of equimolar amounts of two polypeptide chains of Mr 48,000 and 38,000. Determination of the molecular weight of the native factor gave a value which is consistent with a Mr of 86,000 indicating that the factor is composed of one Mr 48,000 and one Mr 38,000 polypeptide. The purified factor exhibited all the binding activities characteristic of eIF-2. The factor formed ternary complexes with Met-tRNAfMet and GTP; it bound GDP to form a binary complex; and it also possessed the property of binding a wide variety of RNA species, including reoviral mRNA, phage T3 mRNA, rRNAs, and tRNA. Furthermore, the ternary complex formed by purified eIF-2 interacted with the 40S ribosomal subunit in the presence of AUG codon to form a 40S initiation complex. These results indicate that all binding activities attributed to eIF-2 are contained in the 48,000- and 38,000-dalton polypeptides.

Animals↗
PubMed1979

Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination.

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.

Adenosine Triphosphatases↗
PubMed1979

Hormonal control of gene expression: differential activation of rat bone marrow RNA polymerases by erythropoietin and testosterone.

Hormones play a role in the regulation of gene expression by inducing changes in enzyme patterns in target cells mediated by the synthesis of specific RNA molecules. Erythropoiesis has been used as a system for studying the molecular mechanism of regulation of gene action by means of two hormones: erythropoietin and testosterone. Experiments designed to correlate the biochemical action of both hormones on rat marrow cells are herein reported. Both factors seems to act at different biochemical and citological levels. Erythropoietin triggers the erythropoietic process acting on the erythropoietin sensitive cells (ESC), in which the hormone induces the synthesis of a high molecular weight RNA, which is the precursor of a functional 9 S messenger RNA. Testosterone seems to act on polychromatophilic erythroblasts, in which the synthesis of ribosomal RNA or its precursor is stimulated. The steroid enhances the nuclear ribonuclease activity, which could represent a control mechanism for the processing (maturation) of high molecular weight RNAs. The incorporation of 3H-GTP and 3H-UTP into RNA by isolated rat bone marrow nuclei is stimulated by erythropoietin and testosterone. Using alpha-amanitine and different ionic strength conditions it was found that erythropoietin enhances preferentially RNA polymerase II activity while testosterone increases RNA polymerase I activity. It is postulated that erythropoietin and testosterone act synergically to create the biochemical machinery for hemoglobin synthesis, the macromolecule that characterizes the erythropoietic process.

Amanitins↗
PubMedDate not supplied

Is sequence conservation in interferons due to selection for functional proteins?

The human alpha-interferon (IFN-alpha) gene family consists of at least 14 potentially functional non-allelic members; the amino acid sequences they encode differ from each other by up to approximately 20% of their residues. Human IFN-beta, which is encoded by a single gene, is distantly related to the IFN-alpha family; it differs in 67% of its residues from IFN-alpha 2. There is considerable evidence that IFN-alpha and -beta compete for the same receptors on their target cells. Comparison of 14 non-allelic human IFN-alpha sequences and the IFN-beta sequence has revealed that 37 of 166 residues are completely conserved and that several of these are arranged in clusters, for example at positions 29-33, 47-50 and 136-150. It is commonly held that evolutionary conservation of amino acids indicates that the residues in question are essential for function. To test this hypothesis in the case of IFNs, we have introduced single site-directed point mutations into the strictly conserved codons 48 and 49 of the IFN-alpha 2 gene which form part of the longest uninterrupted cluster (position 47-50). We report here that the mutant proteins, containing Tyr, Ser and Cys instead of Phe48, or His instead of Gln49, have biological activities indistinguishable from those of wild-type IFN-alpha. In addition, when Glu62, a residue conserved in all known alpha and beta IFNs of man, mouse and cattle, was replaced by Lys, antiviral activity remained unchanged.

Amino Acid Sequence↗