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L R Yarbrough

Publications and source records attributed to L R Yarbrough.

At least 37 records · Page 2Linked to original sources
PubMed1988

Effect of nitrogenous compounds on nitrogenase gene expression in anaerobic cultures of Anabaena variabilis.

The effects of several organic and inorganic nitrogen compounds on nitrogenase mRNA and enzyme activity levels were examined in anaerobic cultures of Anabaena variabilis 29413. Even low concentrations of exogenous ammonia (20 microM) prevented nitrogenase gene expression. Nitrate, in contrast, had little effect, even at very high concentrations. Neither compound had a significant direct effect on existing enzyme activity. The amino acids glutamine and glutamate did not repress nif gene expression. Methionine sulfoximine, but not 7-azatryptophan, was shown to eliminate the repressive effect of ammonia, and this action occurred at the mRNA level. Low concentrations of carbamyl phosphate caused a rapid decrease in nitrogenase mRNA levels. These results are consistent with the ideas that nif gene regulation in Anabaena spp. occurs primarily at the mRNA level and that ammonia, and possibly also glutamine and glutamate, is not the immediate effector of regulation.

Ammonia↗
PubMed1988

Structure and regulation of genes encoding phycocyanin and allophycocyanin from Anabaena variabilis ATCC 29413.

Gene clones encoding phycocyanin and allophycocyanin were isolated from an Anabaena variabilis ATCC 29413-Charon 30 library by using the phycocyanin (cpc) genes of Agmenellum quadruplicatum and the allophycocyanin (apc) genes of Cyanophora paradoxa as heterologous probes. The A. variabilis cpcA and cpcB genes occur together in the genome, as do the apcA and apcB genes; the two sets of genes are not closely linked, however. The cpc and apc genes appear to be present in only one copy per genome. DNA-RNA hybridization analysis showed that expression of the cpc and apc genes is greatly decreased during nitrogen starvation; within 1 h no cpc or apc mRNA could be detected. The source of nitrogen for growth did not influence expression of the genes; vegetative cells from nitrogen-fixing and ammonia-grown cultures had approximately the same levels of cpc and apc mRNAs. Heterocysts had less than 5% as much cpc mRNA as vegetative cells from nitrogen-fixing cultures. Northern hybridization (RNA blot) analysis showed that the cpc genes are transcribed to give an abundant 1.4-kilobase (kb) RNA as well as two less prominent 3.8- and 2.6-kb species. The apc genes gave rise to two transcripts, a 1.4-kb predominant RNA and a minor 1.75-kb form.

Cyanobacteria↗
PubMed1987

Expression of the alpha and beta tubulin genes of the African trypanosome in Escherichia coli.

The African trypanosome, Trypanosoma brucei, contains multiple genes for both alpha- and beta-tubulins, which code for similar if not identical proteins. Studies of the structure and function of trypanosome microtubules have been limited due to the difficulties in obtaining sufficient amounts of purified tubulin. To produce large amounts of purified tubulin for studies of structure and function and to begin developing a system for producing systematic alterations of tubulin structure we have cloned and expressed the alpha- and beta-tubulin genes of T. brucei in Escherichia coli to produce the unfused proteins. Controlled high-level expression of both alpha- and beta-tubulin was achieved using a plasmid vector, pOTS, in which expression is controlled by phage lambda promoter/operator and a temperature-sensitive lambda repressor. The tubulins produced are insoluble, as has been found for many other proteins expressed to high levels in E. coli; they are readily purified to near homogeneity by chromatography on DEAE-cellulose in 7 M urea. N-terminal analysis of the purified proteins indicates that they are initiated correctly and that the N-formyl group is removed from the initiating methionine. This factor will probably prove important in the reconstitution of biologically active tubulin.

Animals↗
PubMed1985

Kinetics of interaction of 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin.

The binding of the guanine nucleotide analogue 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (S6-GTP) to tubulin from which the associated proteins and exchangeably bound nucleotide have been removed produces about a 15% decrease in intrinsic tubulin fluorescence. Using a fluorescence stopped-flow technique, we have examined the kinetics and mechanism of this process. Analysis of the data reveals that the binding is complex, involving at least one conformational change subsequent to nucleotide binding. The bimolecular association rate constant for binding of S6-GTP to tubulin is approximately 6 X 10(5) M-1 s-1, suggesting that the orientation requirements are stringent. The kinetic parameters for dissociation of GDP, S6-GTP, and S6-GDP from the exchangeable nucleotide binding site have also been determined. S6-GDP and GDP were found to have comparable rates of dissociation; S6-GTP dissociated approximately twice as slowly as either GDP or S6-GDP. Glycerol produces a significant decrease in the rates of nucleotide dissociation. The mechanism whereby glycerol produces such an effect is not known; however, it may involve slight changes in the conformation of the tubulin protomer.

Animals↗
PubMed1985

Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense:nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins.

Most tubulin genes of the African trypanosome Trypanosoma rhodesiense are contained in 3.7-kb tandemly repeating units. One member of the 3.7-kb repeat family has been isolated from a T. rhodesiense genomic library, cloned, and sequenced. The 3646-bp fragment contains a complete alpha-tubulin gene and portions of two beta-tubulin genes. No introns are present. The genes are separated by 634- and 333-bp intergenic regions, which lack typical eukaryotic promoter and poly(A) signal sequences. However, both intergenic regions exhibit some structural similarity with sequences proposed to be involved in transcription termination and poly(A) addition in yeast. The 634-bp intergenic region shows homology to the "mini-exon" sequence associated with variable surface glycoprotein (VSG) and other trypanosome mRNAs. A comparable sequence is not found in the 333-bp intergenic region. T. rhodesiense alpha and beta-tubulins exhibit about 84-85% amino acid (aa) sequence homology with tubulins of mammals; the genes show about 74-75% nucleotide sequence homology. The alpha-tubulin contains 451 aa and the beta tubulin 442 aa; both have tyrosine as the C-terminal aa.

Amino Acid Sequence↗
PubMed1984

Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects.

In the presence of glycerol, the thionucleotide 2-amino-6-mercapto-9-ribofuranosyl purine 5'-triphosphate (S6-GTP) promotes the assembly of 6 S tubulin to form microtubules. Microtubules assembled with this analog show normal stability properties. In the absence of glycerol, few microtubules are formed with S6-GTP; however, many twisted ribbons are evident. Binding of S6-GTP to tubulin from which the associated proteins and exchangeable nucleotide have been removed (Tu(-] produces about a 16% quenching of intrinsic tubulin fluorescence. Fluorescence titrations indicate an apparent Kd for the tubulin S6-GTP complex of about 3 X 10(-8)M. Binding of S6-GTP to Tu(-) also produces a change in its absorption spectrum. The observed difference spectrum has a maximum at 350 nm and negative extrema at 323 and 338 nm. This suggests that the environment of the thioguanine ring is relatively hydrophobic. Competitive displacement studies yield apparent Kd values of about 1.7 X 10(-8)M for GTP and 8.3 X 10(-8) M for GDP. The changes in absorbance and fluorescence which accompany binding provide an excellent approach to the study of the kinetics and mechanisms of nucleotide binding as well as studies of the kinetics of displacement of GTP, GDP, and their analogs.

Animals↗
PubMed1980

Stacking interactions in fluorescent nucleotide analogs containing 1-aminonaphthalene-5-sulfonate at the phosphoryl terminus.

The conformational properties of nucleotides containing the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond have been examined. Measurements of fluorescence excitation spectra show that energy absorbed by adenine in the ATP analog is transferred to the naphthalene moiety with an efficiency of approximately 43%. In nonpolar solvents transfer is almost eliminated. The fluorescence intensity of the UTP analog is quenched. Measurements of quantum yield and excited state lifetime show that this occurs by both dynamic and static mechanisms. Thus, fluorescence studies show that both the purine and pyrimidine analogs exist in an equilibrium mixture of stacked and unstacked forms. NMR studies show that the base and ribose protons of the ATP and UTP analogs are shifted upfield by about 0.2 to 0.3 ppm, presumably due to ring current effects produced by stacking interactions. Phosphorus NMR spectra of the ATP analog are generally similar to spectra of unmodified ATP. The strong dependence on conformation of the fluorescence of the pyrimidine analogs may prove useful in studies of protein-nucleotide interactions.

Adenosine Triphosphate↗
PubMed1979

Spectroscopic techniques for study of phosphodiester bond formation by Escherichia coli RNA polymerase.

Nucleotides containing the fluorophore 1-aminonaphthalene-5-sulfonate attached to the gamma phosphoryl group via a phosphoamidate bond are excellent substrates for Escherichia coli DNA-dependent RNA polymerase. Cleavage of the alpha-beta-phosphoryl bond produces significant changes in both absorption and fluorescence spectra. These alterations provide a sensitive and precise means for continuous monitoring of transcription. Under appropriate conditions one can detect the utilization of less than 1 nmol of nucleotide. Since the spectroscopic techniques measure nucleotide utilization they can be used in conjunction with measurements of incorporation of radiolabeled precursor such as [3H]UTP into acid-insoluble material to determine whether significant amounts of acid-soluble oligonucleotides are formed.

DNA-Directed RNA Polymerases↗
PubMed1979

Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases.

A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.

DNA-Directed RNA Polymerases↗
PubMed1976

Conformational transition of Escherichia coli RNA polymerase induced by the interaction of sigma subunit with core enzyme.

The isolated sigma subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(1-pyrene)maleimide. The labeled sigma subunit (PM-sigma) still retained its biological activity in stimulating transcription of T7 DNA by core enzyme. When a stoichiometric amount of core enzyme was added to a solution of PM-sigma, there was a decrease in fluorescence intensity without shifts in emission maxima of PM-sigma. The kinetics of the interaction between the sigma subunit and core enzyme was investigated with the stopped-flow technique by monitoring the fluorescence quenching. A biphasic change of fluorescence intensity with respect to time was observed when PM-sigma was rapidly mixed with an excess of core enzyme. The kinetic data can be analyzed in terms of a mechanism in which a fast bimolecular binding of sigma to core enzyme is followed by a relatively slow isomerization of the holoenzyme formed. From the best-fit kinetic parameters, an overall binding constant of less than or equal to 3X10(-10)M was estimated for the PM-sigma core complex, in agreement with that obtained by the fluorimetric titration. In addition, we have studied the effect of temperature on the rate constant associated with the conformational change of the holoenzyme, which shows a temperature transition around 20 degrees C. The nonlinear Arrhenius plot obtained implies that the conformational transition is complex and may be composed of several processes. The activation energy for the "overall" conformational change was estimated to be 6.7 kcal/mol. The kinetic evidence for the conformational transition of holoenzyme induced by the interactions of sigma subunit with core enzyme presented here further supports the proposition that the sigma subunit acts on core enzyme to trap a unique conformation of RNA polymerase which recognizes the proper promoters and initiates the synthesis of specific RNA chains.

DNA-Directed RNA Polymerases↗
PubMed1976

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.

Binding Sites↗
PubMed1976

Molecular mechanism of the rifampicin -RNA polymerase interaction.

Equilibrium and kinetic studies of the interaction of rifampicin with RNA polymerase of Escherichia coli were performed by exploiting the quenching of intrinsic fluorescence of the protein by the drug. Fluorimetric titrations show that rifampicin binds stoichiometrically to the core and holoenzyme with an apparent Kd of less than or equal to 3 x 10(-9) M. Neither the addition of template nor the formation of the initiation complex in the presence of dinucleotide and nucleoside triphosphate prevents the rifampicin-enzyme interaction. Although the equilibrium binding constant for the rifampicin-RNA polymerase complex is about the same for the core and holoenzyme and the holoenzyme-T7 DNA complex, stopped-flow studies indicate that the rates at which rifampicin interacts with these enzyme forms are different. In all three cases, the kinetic data can be interpreted in terms of a mechanism in which the rapid bimolecular binding of rifampicin to RNA polymerase is followed by a relatively slow isomerization of the drug enzyme complex: (See article). While the values of dissociation constant K1 = (k-1/k1), for the first binary complex (ER) are similar, the rate constant for the forward isomerization, k2, decrease in the order of core enzyme greater than holoenzyme greater than the holoenzyme-T7 DNA complex. The fact that this order is parallel to the relative rates of inactivation of the enzymes and the enzyme-DNA complex suggests that the inactivation may be due to the rifampicin-induced isomerization (conformational change) of the enzyme. This is supported by our observations that an enzyme complex which is in the process of elongating RNA chains can still bind rifampicin, although the enzyme activity is not inhibited by such binding. The values of overall binding constants calculated from the kinetic parameters, 1-2 x 10(-9) M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and Ki determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicin-resistant RNA synthesis and the recent finding that rifampicin only partly inhibits the formation of the first phosphodiester bond in an abortive initiation of RNA chains are consistent with our kinetic mechansim, i.e., the existence of two forms of the rifampicin-RNA polymerase complex, only one of which is able to initiate the RNA chains.

Binding Sites↗
PubMed1976

Spatial relationship of the sigma subunit and the rifampicin binding site in RNA polymerase of Escherichia coli.

sigma subunit of Escherichia coli RNA polymerase is known to stimulate specific RNA chain initiation. Rifampicin, an inhibitor of RNA chain initiation, binds to a single site on the beta subunit of RNA polymerase. We have used the fluorescence energy transfer technique to deduce proximity relationships of sigma subunit and rifampicin binding site on the enzyme. Isolated sigma subunit was covalently labeled with fluorescent donors in two ways: specific labeling of a single sulfhydryl residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate (1,5-I-AENS) and nonspecific labeling on the surface of the protein with dansyl chloride (Dns-Cl) adsorbed on Celite. The labeled sigma subunits were biologically active and formed a stoichiometric complex with core polymerase. The efficiency of energy transfer was obtained from the fluorescence intensity and the excited-state lifetime of the sigma-labeled holoenzyme in the presence and absence of rifampicin, which served as an energy acceptor. The transfer efficiency (2%) from AENS to rifampicin placed AENS somewhere between 42 and 85 A away from the rifampicin binding site. The rotational mobility of the donor was determined by nanosecond fluorescence depolarization spectroscopy, while the acceptor orientation was assumed to be fixed at some unknown angle. The efficiency measured for energy transfer from Dns to rifampicin was 10% in the presence of 0.2 M KCl. The distance from the surface of sigma subunit to the rifampicin binding site was calculated to be 27--38 A for a model having a randomly distributed and oriented array of donors on the surface of a spherical sigma subunit of 31-A radius. Our results indicate that rifampicin does not inhibit the initiation of transcription by RNA polymerase through a direct interaction with sigma subunit. In addition, energy transfer measurements under low salt conditions suggest that in RNA polymerase dimer the two rifampicin binding sites are symmetric with respect to each sigma subunit.

Binding Sites↗
PubMed1975

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

Studies of RNA chain initiation have suggested that the sigma subunit of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) is released from the enzyme-template complex during transcription and may be reused by another core polymerase. Nanosecond fluorescence depolarization spectroscopy was used to follow the sigma cycle. Isolated sigma subunit labeled with the fluorescent probe dansyl (DNS) chloride bound stoichiometrically to core polymerase and stimulated transcription of phage T7 DNA to the same extent as did unlabeled sigma. DNS-sigma showed an exponential fluorescence anisotropy decay corresponding to a rotational correlation time of about 100 nsec. This value was unaffected by addition of T7 DNA, but increased about 6-fold when core polymerase was added, and increased further when T7 DNA was added. Such increases are expected for the formation of molecular complexes. Using the anisotropy decays for free DNS-sigma and DNS-sigma-core enzyme bound to T7 DNA, we calculated theoretical decay curves for various mixtures of free and bound sigma. Comparison of the observed anisotropy decay with the calculated curves indicated that about 55% of DNA-sigma was released from the enzyme-T7 DNA complex in the presence of four nucleoside triphosphates under low salt conditions. Sigma release did not occur if rifampicin was added prior to addition of four nucleoside triphosphates or if only three nucleoside triphosphates were present. After sigma was released, addition of core polymerase with rifampicin reduced the free sigma to less than 15%, indicating that the released sigma was accessible to the added core enzyme. Thus these studies have provided physical evidence for the sigma cycle during in vitro transcription.

Binding Sites↗