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R Sousa

Publications and source records attributed to R Sousa.

At least 55 records · Page 3Linked to original sources
PubMed1997

A model for the mechanism of polymerase translocation.

A general mechanism for polymerase translocation is elaborated. The central feature of this mechanism is that a rapid translocational equilibrium is established after each cycle of nucleoside monophosphate incorporation such that the polymerase distributes itself by diffusional sliding between all accessible positions on the template with relative occupancy determined by relative free energy. While alternative models for translocation have not been fully developed, much of the language currently used to describe this step suggests an active mechanism coupled to conformational transitions in the polymerase. For example, a recent study of force generation by Escherichia coli RNA polymerase during transcription suggests that it is a mechanoenzyme analogous to kinesin of myosin motor proteins. While the proposed mechanism does not rule out conformational transitions during polymerase translocation, it suggests that they may be unnecessary and that translocation can be explained in terms of the affinity of the active site for nucleoside triphosphate and the relative free energies of the polymerase bound at different positions on the template. This mechanism makes specific predictions which are borne out experimentally with polymerases as distinct as E. coli DNAP I, phage T7 RNAP, and E. coli RNAP.

Bacteriophage T7↗
PubMed1996

Structural and mechanistic relationships between nucleic acid polymerases.

A superfamily of nucleic acid polymerases that includes the pol I and pol alpha classes of DNA-directed DNA polymerases, mitochondrial and phage DNA-directed RNA polymerases, and most RNA-directed polymerases may be defined on the basis of the occurrence of conserved sequence motifs and tertiary structure similarities between HIV-1 reverse transcriptase, DNA polymerase I and T7 RNA polymerase. Although sequence or structural similarities do not yet justify inclusion of the multi-subunit DNA-directed RNA polymerases in this superfamily, mechanistic similarities suggest a deep relationship between these and the simpler T7-like RNA polymerases.

Amino Acid Sequence↗
PubMed1995

Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed.

When in Escherichia coli the host RNA polymerase is replaced by the 8-fold faster bacteriophage T7 enzyme for transcription of the lacZ gene, the beta-galactosidase yield per transcript drops as a result of transcript destabilization. We have measured the beta-galactosidase yield per transcript from T7 RNA polymerase mutants that exhibit a reduced elongation speed in vitro. Aside from very slow mutants that were not sufficiently processive to transcribe the lacZ gene, the lower the polymerase speed, the higher the beta-galactosidase yield per transcript. In particular, a mutant which was 2.7-fold slower than the wild-type enzyme yielded 3.4- to 4.6-fold more beta-galactosidase per transcript. These differences in yield vanished in the presence of the rne-50 mutation and therefore reflect the unequal sensitivity of the transcripts to RNase E. We propose that the instability of the T7 RNA polymerase transcripts stems from the unmasking of an RNase E-sensitive site(s) between the polymerase and the leading ribosome: the faster the polymerase, the longer the lag between the synthesis of this site(s) and its shielding by ribosomes, and the lower the transcript stability.

Blotting, Northern↗
PubMed1995

A mutant T7 RNA polymerase as a DNA polymerase.

We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non-conservative mutations of this tyrosine also reduce discrimination between deoxyribo- and ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction.

Bacteriophage T7↗
PubMed1995

Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization.

A protein preparation to be used for crystallization should be homogeneous and should remain so throughout the course of a prolonged crystallization experiment. General methods for preparation of pure proteins and for prevention of their covalent modification (through proteolysis, sulfhydryl oxidation, etc.) during prolonged incubation are well known. Crystallographers are less aware of general methods for stabilization of proteins against non-covalent modifications (partial denaturation, heterogeneous aggregation) which can also introduce structural heterogeneity into a protein preparation. Related to this issue are methods to suppress protein conformational flexibility which can be a source of dynamic structural heterogeneity and which presents an entropic barrier to crystallization. However, for many years agents which stabilize protein structure have been described in the biochemical literature. Recently the most widely used of these structure-stabilizing agents, glycerol, was used to crystallize T7 RNA polymerase. The observation that this compound has general structure-stabilizing effects and that it was essential for crystallization of at least this one protein led to the suggestion that it might be generally useful in crystallizing flexible proteins and in inducing order in disordered segments of crystalline proteins. Subsequently, glycerol was used with good effect in the crystallization of a number of proteins. Other recent results suggest that soaking crystals in solutions containing glycerol can have 'structure-ordering' effects on the crystalline protein. These observations support the utility of glycerol in protein crystallization and suggest that the information in the biochemical literature on protein structure-stabilizing agents will find useful application in the field of protein crystal growth.

Journal Article↗
PubMed1995

Inactivation of genes encoding subunits of the peripheral and membrane arms of neurospora mitochondrial complex I and effects on enzyme assembly.

We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly.

Blotting, Southern↗
PubMed1994

The thumb's knuckle. Flexibility in the thumb subdomain of T7 RNA polymerase is revealed by the structure of a chimeric T7/T3 RNA polymerase.

We have solved the structure of a chimeric T7/T3 RNA polymerase (RNAP) in an orthorhombic crystal by molecular replacement with the T7 RNAP structure determined from a monoclinic crystal. The structure of the protruding "thumb" subdomain of the polymerase appears very different in these two crystals apparently because of differences in packing contacts made by the thumb subdomain. These observations support the proposal that the thumb subdomain is flexible and can wrap around bound template to obstruct polymerase: template dissociation during processive synthesis.

Amino Acid Sequence↗
PubMed1994

The thumb subdomain of T7 RNA polymerase functions to stabilize the ternary complex during processive transcription.

To examine the function of the thumb subdomain in bacteriophage T7 RNA polymerase we constructed a set of deletion mutants within this subdomain. These mutants exhibited reduced processivity during the processive, but not the abortive, stage of transcription. Reduced processivity was found to be due primarily to an increase in the processive ternary complex dissociation rate (destabilization of the processive ternary complex). The destabilization of the ternary complex does not appear to be due to a decrease in the affinity of the polymerase for the nascent RNA. These observations support the proposal that the thumb subdomain functions to stabilize the processive ternary complex during the processive (but not the abortive) stage of transcription, probably by wrapping around the template to prevent polymerase dissociation.

Binding Sites↗
PubMed1994

Characterization of a set of T7 RNA polymerase active site mutants.

We have evaluated the elongation rates, processivities, and abortive transcription characteristics of a set of T7 RNA polymerase mutants that map to the polymerase active site. The effects of these mutations on transcription are complex: they cause decreases in activity and processivity during both the processive and abortive phases of transcription and exhibit disproportionate decreases in activity and processivity on poly(dA).poly(dT) or poly(dT) versus poly(dG).poly(dC) templates. They also exhibit an increase in the proportion of slippage dependent poly(G) transcript synthesis during the initial stages of transcription. It is shown that these multiple, distinct effects on transcription can be attributed to decreases in the mutant enzymes' phosphodiester bond formation rates. Estimates of the decreases in these rates are derived from the measured transcript elongation rates and processivities of the mutant enzymes.

Binding Sites↗
PubMed1993

Crystal structure of bacteriophage T7 RNA polymerase at 3.3 A resolution.

The crystal structure of T7 RNA polymerase reveals a molecule organized around a cleft that can accommodate a double-stranded DNA template. A portion (approximately 45%) of the molecule displays extensive structural homology to the polymerase domain of Klenow fragment and more limited homology to the human immunodeficiency virus HIV-1 reverse transcriptase. A comparison of the structures and sequences of these polymerases identifies structural elements that may be responsible for discriminating between ribonucleotide and deoxyribonucleotide substrates, and RNA and DNA templates. The relative locations of the catalytic site and a specific promoter recognition residue allow the orientation of the polymerase on the template to be defined.

Bacteriophage T7↗
PubMed1993

[Multiple hepatic-splenic abscesses. A favorable result with antibiotic treatment].

The authors report a case of multiple and spleen pyogenic abscesses caused by Peptostreptococcus spp., in a patient who recently underwent laparotomy due to duodenal perforation. He was successfully treated by means of intensive and extended antibiotherapy. We would like to stress the importance of an early and etiological diagnosis performed by percutaneous puncture and bacteriological study, in order to start a selective antibiotherapy. Other therapeutical possibilities are discussed.

Abscess↗
PubMed1992

Isolation and characterization of mutant bacteriophage T7 RNA polymerases.

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.

Catalysis↗
PubMed1992

Model for the mechanism of bacteriophage T7 RNAP transcription initiation and termination.

Characterization of a mutant T7 RNA polymerase (RNAP) that is active on non-promoter templates but has lost the ability to selectively utilize the T7 promoter led to the finding that wild-type T7 RNAP initiates transcription at a high rate on non-promoter templates but that most (approximately 90%) of these initiation events lead to synthesis of dinucleotides only. The anomalously high activity of T7 RNAP on poly(dC) templates (relative to other non-promoter templates) is due to a reduction in the rate of transcription abortion after dinucleotide synthesis rather than an increase in initiation. Evidence is presented that the transition from abortive to processive transcription is associated with a conformational change in T7 RNAP. The stability of the nascent chain in a ternary complex is shown to increase with increasing chain length in the 2 to 14 base range even when the size of the complementary RNA-DNA hybrid remains constant and small (2 to 3 base-pairs). Two mutant polymerases that show increased release of transcripts during abortive transcription and a proteolytically nicked polymerase that exhibits reduced RNA binding are shown to have reduced ability to read-through a T7 RNAP hairpin U-stretch transcription terminator. Single-stranded nucleic acids are shown to bind more tightly than double-stranded nucleic acids to T7 RNAP. These observations and a large set of published studies on T7 RNAP structure and mechanism are accommodated in a relatively simple model of T7 RNAP transcription initiation and termination in which a T7 RNAP that has initiated transcription is proposed to be capable of assuming two functionally distinct conformations: an abortive conformer characterized by a loose association with the nascent RNA and an inability to translocate along the template; and a processive conformer characterized by the stable retention of the nascent RNA and the ability to process stably along the template. The equilibrium between these two conformations is shifted towards the processive form when the nascent chain binds at a site located at least partly on the T7 RNAP N-terminal domain. The interaction requires that the RNA be more than approximately nine bases and this RNAP-RNA interaction plays a primary role in retaining the RNA within the ternary complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1992

Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure.

In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP. These observations support the proposal that these polymerase active sites have similar three-dimensional structures.

Amino Acid Sequence↗
PubMed1992

Characterization of a novel synapse-specific protein. I. Developmental expression and cellular localization of the F1-20 protein and mRNA.

A molecular description of the nerve terminal will be required to understand synaptic function fully. The goals of this study were to contribute toward such a description by characterizing a novel synapse-specific protein. A monoclonal antibody library was screened for antibodies to synaptic proteins. The antibodies were then used to isolate cDNA clones by expression screening. Here we report a detailed characterization of the protein reactive with monoclonal antibody F1-20. Immunohistochemical and biochemical analyses revealed that the F1-20 protein is synapse associated. Western blot analyses revealed that the F1-20 protein is a brain-specific polypeptide with an apparent molecular weight on SDS-PAGE of 190,000 Da. Northern blot analyses indicated that probes generated from an F1-20 cDNA clone hybridize to a single brain-specific mRNA of approximately 4.8 kilobases. In situ hybridization experiments demonstrated that F1-20 mRNA expression is neuronal specific. Northern and Western blot analyses indicated that F1-20 mRNA levels increase abruptly at postnatal day 4 and protein levels increase abruptly at postnatal day 7. This corresponds to a period of active synaptogenesis and synaptic maturation in the mouse CNS. We characterized the neuroanatomical distribution of the F1-20 protein by immunohistochemistry, and of the F1-20 mRNA by in situ hybridization. We found that the F1-20 mRNA and protein are expressed nonuniformly in brain. Variation in the expression of the F1-20 protein is complex and reveals patterns also exhibited by probes directed against other synapse-associated molecules. The highest levels of F1-20 protein are found in the cortically organized regions of the brain. The highest levels of F1-20 mRNA are found in long-distance projection neurons. There is also variation in the expression of F1-20 mRNA between different classes of large output neuron, as well as extensive variation in the expression of F1-20 mRNA between different nuclear groups.

Aging↗