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S A Benner

Publications and source records attributed to S A Benner.

At least 91 records · Page 5Linked to original sources
PubMed1991

Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron.

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.

Animals↗
PubMed1991

RNA world.

Explore the source record for details and available documents.

Biological Evolution↗
PubMed1991

Site-directed mutagenesis of bovine pancreatic ribonuclease: lysine-41 and aspartate-121.

Chemical modification studies suggest that two residues of bovine pancreatic ribonuclease A (RNase A), Lys-41 and Asp-121, are important for catalysis. Three mutants of RNase A have been prepared, two point mutants with Lys-41 altered to Arg-41 and Asp-121 altered to Glu-121, and a double mutant where both residues are altered. The Lys-41 Arg mutant has ca. 2% the catalytic activity (kcat/Km) of the native protein, while the Asp-121Glu mutant has ca. 17% the catalytic activity of the native protein. The double mutant has catalytic activity comparable to the Lys-41Arg mutant.

Amino Acid Sequence↗
PubMed1991

Patterns of divergence in homologous proteins as indicators of secondary and tertiary structure: a prediction of the structure of the catalytic domain of protein kinases.

The secondary structure and elements of tertiary structure have been predicted for the catalytic domain of protein kinases using a method that extracts structural information from the patterns of conservation and variation in an alignment of homologous proteins. The central features of this structural prediction are: (a) the catalytic domains of protein kinases do not incorporate a Rossmann fold; (b) the core of the structure is founded on beta sheets built from pairs of bent antiparallel beta strands; (c) five helices, including an especially long helix (alignment positions 129-152) that lie on the outside of the folded core. These proteins are important in many aspects of metabolic regulation.

Animals↗
PubMed1991

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3'----5')-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable 'module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.

Amino Acid Sequence↗
PubMed1990

Interferon-gamma activates the cleavage of double-stranded RNA by bovine seminal ribonuclease.

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([3H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-gamma (IFN-gamma). Activation is seen with as little as 4-10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-gamma's ability to control cell growth and induce an antiviral state.

Animals↗
PubMed1990

The stereospecificities of seven dehydrogenases from Acholeplasma laidlawii. The simplest historical model that explains dehydrogenase stereospecificity.

Stereospecificities are reported for seven dehydrogenases from Acholeplasma laidlawii, an organism from an evolutionarily distinct branch of life which has not previously been studied from a stereochemical point of view. Three of the activities examined (alcohol dehydrogenase, lactate dehydrogenase, and alanine dehydrogenase) catalyze the transfer of the pro-R (A) hydrogen from NADH. Four other activities (3-hydroxy-3-methylglutaryl-CoA reductase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and NADH oxidase) catalyze the transfer of the pro-S (B) hydrogen from NAD(P)H. The stereospecificity of hydroxymethylglutaryl-CoA reductase is notable because it is the opposite of that of hydroxymethylglutaryl-CoA reductases from yeast and rat. These data are used to derive the simplest historical model capable of explaining available experimental facts.

Acholeplasma laidlawii↗
PubMed1990

The ribonuclease from an extinct bovid ruminant.

The sequence of the ribonuclease from the ancestor of swamp buffalo, river buffalo, and ox, corresponding approximately to Pachyportax latidens, an extinct ruminant known from the fossil record, has been reconstructed using the rule of 'maximum parsimony'. This protein and two sequences that may have been intermediates in the evolution of modern ribonuclease have been constructed in the laboratory by site-directed mutagenesis, and their properties examined.

Amino Acid Sequence↗
PubMed1989

An improved system for expressing pancreatic ribonuclease in Escherichia coli.

An improved method for expressing and purifying bovine pancreatic ribonuclease from a synthetic gene using the lambda promoter controlled by a temperature-sensitive repressor is described. The procedure involves isolation in the presence of a refolding buffer containing oxidized and reduced glutathione, under conditions where RNase can refold, but where proteases presumably do not. Yields are approx. 2 mg purified protein per 1 ferment.

Bacteriophage lambda↗
PubMed1989

The stereospecificity of the ferrous-ion-dependent alcohol dehydrogenase from Zymomonas mobilis.

Alcohol dehydrogenase from Zymomonas mobilis has been found to transfer the pro-R hydrogen of NADH to acetaldehyde. This is the first report of the stereospecificity of a dehydrogenase in the mechanistic and structural class of Fe2+-dependent alcohol dehydrogenases and offers an opportunity to expand mechanistic hypotheses relating stereospecificity, reaction mechanism and reaction thermodynamics in dehydrogenases.

Acetaldehyde↗
PubMed1989

Patterns of divergence in homologous proteins as indicators of tertiary and quaternary structure.

A new approach for extracting conformational information from an alignment of homologous proteins is presented. This approach extracts information from the pattern of sequence divergence in proteins, and considers evolutionary issues, such as functional adaptation and neutral drift, in assigning roles in tertiary structure to residues at specific positions in the alignment. A reliable algorithm is developed for identifying surface residues in a protein. An algorithm is also developed for identifying active site residues; this algorithm can be applied in cases where functional divergence occurs in one subgroup of homologous proteins but not in others. Finally, these algorithms are used to make predictions regarding the quaternary structure of alcohol dehydrogenase from yeast.

Alcohol Dehydrogenase↗
PubMed1989

The return of pancreatic ribonucleases.

A decade after losing favor as an 'uninteresting' digestive enzyme, pancreatic ribonuclease has been found to be homologous to a series of extracellular proteins that may influence tumor cell growth, neurological development and biological differentiation. One surprising outcome of these discoveries has been the confirmation of the hypothesis that extracellular 'communicator RNA' is a messenger important in cell growth and differentiation. The only question is: why wasn't this recognized earlier?

Animals↗
PubMed1989

Modern metabolism as a palimpsest of the RNA world.

An approach is developed for constructing models of ancient organisms using data from metabolic pathways, genetic organization, chemical structure, and enzymatic reaction mechanisms found in contemporary organisms. This approach is illustrated by a partial reconstruction of a model for the "breakthrough organism," the last organism to use RNA as the sole genetically encoded biological catalyst. As reconstructed here, this organism had a complex metabolism that included dehydrogenations, transmethylations, carbon-carbon bond-forming reactions, and an energy metabolism based on phosphate esters. Furthermore, the breakthrough organism probably used DNA to store genetic information, biosynthesized porphyrins, and used terpenes as its major lipid component. This model differs significantly from prevailing models based primarily on genetic data.

Bacteria↗
PubMed1988

Extracellular 'communicator RNA'.

Evidence is presented that RNA molecules exist that act as extracellular messages important in the development of higher organisms.

Extracellular Space↗
PubMed1988

The design of synthetic genes.

Computer programs are described that aid in the design of synthetic genes coding for proteins that are targets of a research program in site directed mutagenesis. These programs "reverse-translate" protein sequences into general nucleic acid sequences (those where codons have not yet been selected), map restriction sites into general DNA sequences, identify points in the synthetic gene where unique restriction sites can be introduced, and assist in the design of genes coding for hybrids and evolutionary intermediates between homologous proteins. Application of these programs therefore facilitates the use of modular mutagenesis to create variants of proteins, and the implementation of evolutionary guidance as a strategy for selecting mutants.

Algorithms↗
PubMed1987

Free energy differences between enzyme bound states.

A theory is presented that describes the free energy difference between the enzyme-substrate (ES) and enzyme-product (EP) complexes that is expected in enzymes optimized for catalytic efficiency. In such enzymes, the free energy drop between ES and EP complexes reflects a portion of the chemical potential difference between substrates and products outside the active site under physiological conditions. Qualitative and quantitative predictions of the model are discussed and compared with experimental data. The controversy over the kinetically optimal free energy profile for an enzymatic reaction operating under constraints set forward by Albery & Knowles (1976) is resolved.

Enzymes↗