Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glu58 is known to participate in phosphodiester transesterification catalyzed by the enzyme RNase T1. For Glu58 RNase T1, an altered mechanism has been proposed in which His40 replaces Glu58 as the base catalyst [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. Glu58Ala Rnase T1 has been cocrystallized with guanosine 2'-monophosphate (2'-GMP). The crystals are of space group P2(1), with one molecule per asymmetric unit (a = 32.44 A, b = 49.64 A, c = 26.09 A, beta = 99.17 degrees). The three-dimensional structure of the enzyme was determined to a nominal resolution of 1.9 A, yielding a crystallographic R factor of 0.178 for all X-ray data. Comparison of this structure with wild-type structures leads to the following conclusions. The minor changes apparent in the tertiary structure can be explained by either the mutation of Glu58 or by the change in the space group. In the active site, the extra space available through the mutation of Glu58 is occupied by the phosphate group (after a reorientation) and by a solvent molecule replacing a carboxylate oxygen of Glu58. This solvent molecule is a candidate for participation in the altered mechanism of this mutant enzyme. Following up on a study of conserved water sites in RNase T1 crystallized in space group P2(1)2(1)2(1) [Malin, R., Zielenkiewicz, P., & Saenger, W. (1991) J. Mol. Biol. 266, 4848-4852], we investigated the hydration structure for four different packing modes of RNase T1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Crystallographic study of Glu58Ala RNase T1 x 2'-guanosine monophosphate at 1.9-A resolution. 790 40

An efficient automatic method has been developed for docking a ligand molecule to a protein molecule. The method can construct energetically favorable docking models, considering specific interactions between the two molecules and conformational flexibility in the ligand. In the first stage of docking, likely binding modes are searched and estimated effectively in terms of hydrogen bonds, together with conformations in part of the ligand structure that includes hydrogen bonding groups. After that part is placed in the protein cavity and is optimized, conformations in the remaining part are also examined systematically. Finally, several stable docking models are obtained after optimization of the position, orientation and conformation of the whole ligand molecule. In all the screening processes, the total potential energy including intra- and intermolecular interaction energy, consisting of van der Waals, electrostatic and hydrogen bonding energies, is used as the index. The characteristics of our docking method are high accuracy of the results, fully automatic generation of models and short computational time. The efficiency of the method was confirmed by four docking trials using two enzyme systems. In two attempts to dock methotrexate to dihydrofolate reductase and 2'-GMP to ribonuclease T1, the exact structures of complexes in crystals were reproduced as the most stable docking models, without any assumptions concerning the binding modes and ligand conformations. The most stable docking models of dihydrofolate and trimethoprim, respectively, to dihydrofolate reductase were also in good agreement with those suggested by experiment. In all test cases, it was shown that our method can accurately predict the correct docking structures, discriminating the correct model from incorrect ones. The efficiency of our method was further tested from the viewpoint of ability to predict the relative stability of the docking structures of two triazine derivatives to dihydrofolate reductase. Our docking method provides a useful tool for rational drug design and investigations of biochemical reaction mechanisms.
J Mol Biol 1994 Oct 21
PMID:Rational automatic search method for stable docking models of protein and ligand. 793 57

The bloodstream forms of the protozoan parasite Trypanosoma brucei lack spectrally detectable cytochromes and satisfy energy requirements mainly by glycolysis. When infected blood is ingested by the tse-tse fly vector, the bloodstream form cells differentiate to procyclic forms that have fully functional mitochondria. Procyclic cells have cyanide-sensitive, cytochrome-mediated electron transport and the full complement of TCA cycle enzymes. The developmental regulation of the cytochrome c reductase complex was examined at the RNA and protein levels. RNase T1 protection studies and Northern blot analyses demonstrated that bloodstream and procyclic form cells constitutively expressed the genes for two nuclear encoded cytochrome c reductase subunits, cytochrome c1 and subunit 4. Polyadenylated transcripts of both genes were present in bloodstream form cells at up to 20% of the procyclic cell levels. These levels were significantly up-regulated sometime after the onset of differentiation to the procyclic form. Despite the presence of subunit mRNAs in bloodstream form cells, subunit proteins were not detected until the cells had been allowed to differentiate in vitro for 6 h. Procyclic cell levels of subunit proteins and holocytochromes were reached by 48 h. Our results suggest that cytochrome c reductase is developmentally regulated at multiple levels, some involving post-transcriptional mechanisms.
Mol Biochem Parasitol 1994 Jun
PMID:Developmental regulation of Trypanosoma brucei cytochrome c reductase during bloodstream to procyclic differentiation. 796 70

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis.
Biochem Mol Biol Int 1994 Aug
PMID:Accurate transcription initiation by RNA polymerase II from Candida utilis. 798 59

Disulfide bonds in a folding protein chain are equivalent to prematurely formed native-like tertiary interactions. We investigated whether the mechanism of protein folding is changed by the presence of disulfide bonds. As a model we used the S54G/P55N-variant of ribonuclease T1, a protein with two disulfide bonds and a single cis proline (Pro39), and we measured both the direct and the proline-limited folding reactions before and after breaking of the disulfide bonds. The folding kinetics were compared under refolding conditions, in the regions of the urea-induced unfolding transitions of the two forms, and under unfolding conditions. The kinetics in the transition regions were analyzed on the basis of a three-species mechanism and all microscopic rate constants of folding and of prolyl isomerization could be determined as a function of the urea concentration from the measured rates and amplitudes. These kinetic analyses indicated that the disulfide bonds can be rather unfavorable for the folding of S54G/P55N-ribonuclease T1. Under strongly native conditions they retard the rate-limiting trans-->cis isomerization of Pro39 because they allow the rapid formation of partially ordered structure prior to the proline-limited refolding reaction. Under unfolding conditions the isomerization of Pro39 is not affected. The direct unfolding and refolding reactions in the transition region of polypeptide chains with correct prolyl isomers are also decelerated when the disulfide bonds are present. These changes in the folding kinetics are possibly related to the decrease in chain flexibility that is caused by the disulfide bonds. A high flexibility is probably important throughout folding, and in the case of ribonuclease T1 a premature locking of tertiary contacts by intact disulfide bonds can interfere unfavorably with both the direct and the proline-limited folding reactions.
J Mol Biol 1994 Jun 24
PMID:Intact disulfide bonds decelerate the folding of ribonuclease T1. 801 91

The cis conformation of the 38-39 peptide bond of ribonuclease T1 is retained after the replacement of cis Pro39 by an alanine residue. This conformation is demonstrated by the presence of a NOESY cross-peak in the NMR spectrum between the C alpha protons of Tyr38 and Ala39 in the Pro39-->Ala variant. The presence of this non-prolyl cis peptide bond explains the retention of the catalytic activity, the strong decrease in stability and the changes in the folding mechanism that were observed after the Pro39-->Ala mutation in ribonuclease T1. We suggest that a cis peptide bond is retained in a protein after the substitution of a cis proline at positions, where a trans bond would destabilize the protein more strongly than a non-prolyl peptide bond in the energetically unfavourable cis conformation.
J Mol Biol 1994 Jul 22
PMID:Generation of a non-prolyl cis peptide bond in ribonuclease T1. 803 56

Alterations in flexibility of monomeric proteins induced by hydrostatic pressure in the predenaturational range (< or = 3 kbar) were probed through the decay kinetics of tryptophan phosphorescence. With apoazurin, ribonuclease T1, wild-type and V67G mutant and phosphoglycerate kinase, pressure effects on the triplet lifetime (tau) and the amplitudes of multicomponent decays emphasize that subtle changes in conformation are ubiquitous. With apoazurin the increase in tau attests to a tightening of the protein core that is enhanced at high temperature. On the contrary, tau decreases with ribonuclease T1, wild-type and mutant, and with phosphoglycerate kinase, indicating that pressure induces a greater flexibility to protein regions in proximity to the surface of the macromolecule. For phosphoglycerate kinase the decrease in tau and the parallel increase in fluorescence intensity and red-shift of the fluorescence spectrum unveil an "unfolding" like transition with midpoint pressures of 1.1 kbar at 5 degrees C and 1.6 kbar at 25 degrees C. Evidence that unfolding of the C-domain of this protein is, however, less than complete is provided by a delta G zero that is about half of that obtained by denaturation in guanidine hydrochloride and also by the ability of this structure to undergo conformational drift. In 70% glycerol, pressure effects on tau of apoazurin are attenuated while for ribonuclease T1 there is a reversal of the tendency with a pronounced increase in tau. With phosphoglycerate kinase glycerol abolishes entirely the "unfolding" transition and all hysteresis effects. A consistent picture of these findings is provided in terms of the location of the probe and of the opposing effects that pressure exerts on protein flexibility by reducing internal cavities and increasing the hydration of the polypeptide.
J Mol Biol 1994 Sep 23
PMID:Pressure effects on protein flexibility monomeric proteins. 808 48

We have reported previously about the cloning of the binase gene in E. coli. In this work, using an original approach named "homolog gene recombination" method (HGR), vectors for binase expression in E. coli have been constructed. Transcription of the binase gene have been directed through either tac-promoter or PR-promoter of bacteriophage lambda under the control of temperature-sensitive CI857 repressor. The last promoter gave the maximum yield of binase, up to 100 mg of protein per litre of heat-induced bacterial culture. The location of the transcription terminator at the 3' terminus of the binase gene raised the expression approximately two times more. A chromatographic method have been developed and applied for the control of binase accumulation in growth medium without measuring the ribonuclease activity.
Mol Biol (Mosk)
PMID:[Superproduction of Bacillus intermedius 7P ribonuclease (binase) in Escherichia coli]. 818 78

The gene for extracellular low molecular weight ribonuclease of Bacillus circulans BCF 247 was cloned. The strain was isolated from permafrost deposits of the Kolyma lowland. The gene for the ribonuclease from Bacillus intermedius (binase) was used as a specific probe. The cloning succeeded only in the E. coli strain producing the inhibitor of ribonuclease form Bacillus amyloliquefaciens. Selected clones secreted the active ribonuclease into the growth media. Deletion derivatives of the parental recombinant plasmid were constructed. The smallest DNA fragment which enclosed a functional ribonuclease gene in E. coli was determined to be 0.6 kb in length.
Mol Biol (Mosk)
PMID:[Cloning of the gene for extracellular Bacillus circulans RNAase]. 818 79

The secondary structure of ribonuclease T1 (RNase T1) in aqueous solution and its temperature-induced structural changes have been investigated by Fourier-transform infrared (FT-IR) spectroscopy. 13 to 14% alpha-helix and 32 to 33% beta-sheet were estimated from the resolution-enhanced FT-IR spectra, in agreement with the crystal structure which indicates 16% alpha-helix and 35% beta-sheet. Specific IR-marker bands are assigned to the different beta-sheet structures, to the slightly bent alpha-helix, and to beta-turn and irregular conformations present in RNase T1. The temperature dependence of the infrared spectra shows that the thermal unfolding and refolding of RNase T1 is fully reversible. This permitted the detailed analysis of structural changes that occur as a function of temperature by evaluating quantitatively the various secondary structure-related amide I band components and some amino acid side-chain vibrations as specific monitors. The secondary structure of RNase T1 is essentially retained in the temperature range between 20 and 50 degrees C. Significant perturbation of protein structure is initiated between 50 and 55 degrees C within regions of beta-sheet structures while the alpha-helix remains virtually intact up to 55 degrees C suggesting a "premelting" of RNase T1. Between 55 and 60 degrees C, a highly co-operative unfolding process is indicated by the simultaneous breakdown of all secondary structure components and by distinct changes of some specific side-chain vibrations. An analysis of the amide I band contour of RNase T1 at 70 degrees C proves that the unfolded state is predominantly, but not completely, irregular or "random coil". Residual, turn-like structures persisting even in the unfolded state are suggested by minor, turn related band components in the amide I region. From IR-spectra collected along a linear temperature gradient, intensity/temperature and frequency/temperature profiles were constructed using some peptide backbone and amino acid side-chain marker bands as local, structure-sensitive monitors. From these profiles individual transition temperatures tm and transition enthalpies delta H (van't Hoff) were calculated. The tm and delta H values revealed a small but distinct hysteresis between repetitive cycles of unfolding and refolding of the protein, suggesting slow refolding kinetics of RNase T1. Furthermore, the various infrared "marker bands" indicate a slightly different response towards temperature increase/decrease for different regions of the protein. The data demonstrate that infrared spectroscopy permits both the detailed analysis of structural changes occurring in a protein as a function of temperature and the determination of thermodynamic parameters characterizing its folded/unfolded state transition.
J Mol Biol 1993 Aug 05
PMID:Secondary structure and temperature-induced unfolding and refolding of ribonuclease T1 in aqueous solution. A Fourier transform infrared spectroscopic study. 835 80


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