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M J Gait

Publications and source records attributed to M J Gait.

At least 37 records · Page 2Linked to original sources
PubMed1996

Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex.

The human immunodeficiency virus type 1 transactivator protein, Tat, stimulates transcriptional elongation from the viral long terminal repeat. To test whether Tat associates directly with activated transcription complexes, we have used the lac repressor protein (LacR) to "trap" elongating RNA polymerases. The arrested transcription complexes were purified by binding biotinylated templates to streptaviridin-coated magnetic beads. Transcription complexes were released from the magnetic beads following cleavage of the templates with restriction enzymes and were immunoblotted with antibodies to Tat, LacR and RNA polymerase II. The Tat protein copurified with RNA polymerase bound to wild-type templates but did not copurify with transcription complexes prepared by using templates carrying mutations in the transactivation response element (TAR) RNA. We conclude that Tat and cellular cofactors become attached to the transcription complex during its transit through TAR.

Base Sequence↗
PubMed1996

Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure.

The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2'-deoxy-nucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.

Base Composition↗
PubMed1996

[Synthesis and properties of mixed ribodeoxyribooligonucleotide duplexes containing an internucleotide trisubstituted pyrophosphate bond].

Fragments of double-stranded RNAs and mixed double-stranded ribo/deoxyribooligonucleotides containing an internucleotide trisubstituted pyrophosphate bond in a predetermined position of the sugar-phosphate backbone were synthesized. The modified internucleotide bond was created between synthetic oligonucleotides by chemical ligation with carbodiimide. The chemical ligation conditions were optimized for a series of duplexes that differed in the content and mutual arrangement of ribo- and deoxyribonucleotide blocks. The yield of oligonucleotides containing the trisubstituted internucleotide pyrophosphate bond was 90% in an all-deoxy duplex and 15-68% in ribooligonucleotide-containing duplexes. The modified duplexes thus obtained are relatively stable in an aqueous solution at pH 5.5-7.5, whereas the modified internucleotide bond is cleaved by amines to form phosphamide derivatives of the corresponding oligonucleotides. Automated solid-phase synthesis of oligonucleotide 3'-ethylphosphates is described.

Base Sequence↗
PubMed1995

Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure.

To find conditions for obtaining diffraction-quality crystals of a hammerhead RNA rapidly and reproducibly, we employed a "double screening" procedure in which we screened six different RNA synthetic constructs against 48 crystallization conditions using a newly devised sparse matrix. We obtained crystals immediately and diffraction-quality crystals of the sixth RNA construct within six months of initiating the screening of additional RNA sequences. The best crystals diffract to 2.9 A resolution when flash-cooled at synchrotron X-ray sources. Solid-support chemical synthesis combined with sparse matrix screening should allow rapid production of diffraction-quality crystals of a variety of small RNAs, reducing the time commitment for initiating such crystallography projects from several years to several months. The synthetic approach also makes introduction of modified bases to prevent self-cleavage and to generate isomorphous heavy-atom derivative crystals a rapid and straightforward process.

Base Sequence↗
PubMed1995

The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription.

The effects of mutations in human immunodeficiency virus type-1 (HIV-1) long terminal repeat on initiation and on Tat-mediated trans-activation were studied using cell-free transcription assays. All the elements that are necessary for efficient transcription initiation in vitro are included in the core promoter. This region contains three tandem Sp1 binding sites, a TATA element and an initiator (INR) sequence. Although the HIV-1 INR element overlaps the trans-activation response region (TAR), it forms an integral part of the promoter. The HIV-1 INR element was characterised in detail using a template that carries a complete HIV-1 promoter and a displaced TAR RNA element. The results demonstrate that the sequence G+1GGTCT is essential for HIV-1 INR function. RNase protection experiments show that Tat acts exclusively to stimulate transcriptional elongation. Mutations in the core promoter elements reduce initiation rates dramatically but do not block Tat activity. For each mutation studied, the total level of transcription in the presence of Tat is proportional to the rate of initiation in the absence of Tat. Furthermore the rate of initiation remains constant in the presence or absence of Tat. We conclude that the elements of the HIV-1 core promoter act in concert to simulate initiation. By contrast, Tat acts independently of the core promoter elements and stimulates elongation. The data strongly suggest that Tat is recruited to the elongating transcription complex during its transit through TAR.

Base Sequence↗
PubMed1995

Purine functional groups in essential residues of the hairpin ribozyme required for catalytic cleavage of RNA.

Synthetic chemistry techniques have been used to study the functional group requirements of the essential purine residues in hairpin ribozyme cleavage. Three-stranded ribozymes were prepared that had functional group deletions or alterations at single purine sites within loops A and B of the hairpin, and the kinetics of cleavage were compared to those of the unmodified ribozyme. Adenosine analogues used were purine riboside and N7-deazaadenosine, and guanosine analogues used were inosine, N7-deazaguanosine, and O6-methylguanosine. In many cases, introduction of one of these analogues caused substantial loss of ribozyme cleavage activity. Most of the impairments of activity were found to be due to changes in kcat rather than in KM. The losses corresponded in magnitude to loss of at least one hydrogen bond, and the results were rationalized in terms of removal of potential cross-strand hydrogen bonds as well as potential hydrogen bonds between loops A and B. A new secondary structure model for loop B was proposed. Finally, the magnesium ion dependence of cleavage was studied for the modified ribozymes and compared to that of the unmodified ribozyme. It is proposed that magnesium binds in the ground state to the N7-positions of G + 1 and A43 and in the transition state to the N7-position at A9. The results provide further evidence for the folding of the two arms of the hairpin so that in the active conformation loops A and B approach closely to form a specific three-dimensional structure with a magnesium ion (or ions) placed between the loops, making contacts in the ground state and in the transition state.

Adenosine↗
PubMed1995

The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription.

The human immunodeficiency virus type 1 (HIV-1) trans-activator protein, Tat, specifically stimulates transcription from the viral long terminal repeat. Tat binds to an RNA stem-loop structure encoded by the trans-activation response region (TAR). To test whether TAR is functional when displaced downstream of the start of transcription, we assayed a series of templates carrying duplicated TAR elements in cell-free transcription systems. When the normally positioned TAR element (TAR-1) is inactivated by mutations in either the Tat binding site or the apical loop sequence, which acts as the binding site for a cellular factor, transactivation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even when placed > 200 nt downstream of TAR-1. TAR complementation experiments have also shown that a functional TAR element requires both an intact Tat binding site and an intact apical loop sequence. For example, if TAR-1 carries a mutation in the loop element it cannot be rescued by a TAR-2 element carrying a mutation in the Tat binding site. Substitution mutations in TAR-1 show that the 5' half of TAR also encodes an essential DNA element which is required for efficient transcription initiation. These results strongly suggest that Tat and cellular cofactors which bind TAR RNA associate with the transcription complex during its transit through TAR.

Base Sequence↗
PubMed1995

Progress in anti-HIV structure-based drug design.

The course of drug development for the treatment of HIV-1 infection and AIDS is being revolutionized by high-resolution structures of essential viral proteins. We survey the impact on drug design of the recently elucidated structural knowledge of two essential enzymes, reverse transcriptase and protease, and three new targets, the viral integrase and the gene regulatory protein-RNA interactions, Tat-TAR and Rev-RRE.

Antiviral Agents↗
PubMed1994

A molecular rheostat. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression.

The complete biologically active human immunodeficiency virus type-1 (HIV-1) rev-response element (RRE) RNA is 351 nucleotides (nt) in length, and includes an extra 58 nt on the 5' end and 59 nt on the 3' end beyond the sites proposed in the original models for the RRE secondary structure. The extra sequences are able to form a duplex structure which extends Stem I. The presence of an elongated Stem I structure in the RRE RNA was confirmed by nuclease mapping experiments. Nuclease protection experiments have shown that rev binds to restricted regions of the RRE, including the high affinity site located at the base of Stem IIb and along the length of the Stem I region. The three large stem-loop structures which protrude from Stem I and Stem IIb (Stems IIc, III+IV and V) remain accessible to nucleases even in the presence of a large excess of protein. Gel-retardation experiments show that the truncations of Stem I reduced the total number of rev molecules that can bind co-operatively and with high affinity to the RRE RNA. To test whether the elongated Stem I structure is required for maximal rev activity, a series of truncations which progressively reduced the length of Stem I was introduced into an HIV-1 derived reporter plasmid. In the presence of rev and a functional RRE, there is an increase in the levels of gag and env mRNA in the cytoplasm and a decrease in levels of tat and rev mRNAs. Each of the truncations in Stem I reduced the rev responses, with the longest truncations producing the greatest losses of activity. The data suggest that the RRE acts as a "molecular rheostat" designed to detect rev levels during the early stages of the HIV growth cycle.

Base Sequence↗
PubMed1994

Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.

The HIV-1 regulatory proteins tat and rev are both RNA binding proteins which recognize sequences in duplex RNA which are close to structural distortions. Here we identify phosphate contacts which are critical for each binding reaction by use of a new method. Model RNA binding sites are constructed carrying substitutions of individual phosphodiesters by uncharged methylphosphonate derivatives isolated separately as Rp and Sp diastereoisomers and tested for protein binding by competition assays. In the binding of tat to the trans-activation response region (TAR), three phosphates, P21 and P22 which are adjacent to the U-rich bulge and P40 on the opposite strand, are essential and in each case both isomers inhibit binding. Similarly, in the interaction between the HIV-1 rev protein and the rev-responsive element (RRE) both methylphosphonate isomers at P103, P104, P124 and P125 interfere with rev binding. At P106, only the Rp methylphosphonate isomer is impaired in rev binding ability and it is proposed that the Rp oxygen is hydrogen-bonded to an uncharged amino acid or to a main chain hydrogen atom. Synthetic chemistry techniques also provide evidence for the conformations of non-Watson-Crick G106:G129 and G105:A131 base-pairs in the RRE 'bubble' structure upon rev binding. Almost all functional groups on the 5 bulged residues in the bubble have been ruled out as sites of contact with rev but, by contrast, the N7-positions of each G residue in the flanking base-pairs are identified as sites of likely hydrogen-bonding to rev. The results show that both tat and rev recognize the major groove of distorted RNA helixes and that both proteins make specific contacts with phosphates which are displaced from the sites of base-pair contact.

Base Composition↗
PubMed1994

Synthetic oligoribonucleotides carrying site-specific modifications for RNA structure-function analysis.

Synthetic oligoribonucleotides have become increasingly valuable in studies of RNA structure and function. A range of nucleotide analogues is available which carry modifications in the base, sugar or phosphate moieties. Such analogues have been incorporated into synthetic RNA structures to eliminate or alter individual functional groups in the RNA which potentially can take part in hydrogen-bonding or other non-covalent interactions. Comparisons of the properties of the modified RNAs with unmodified RNA models allow conclusions to be drawn concerning the importance or otherwise of specific functional groups within the RNA. These methods have been applied to studies of RNA interactions with proteins, RNA catalysis and RNA structure.

Base Sequence↗
PubMed1993

The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.

The synthesis is described of oligoribonucleotides containing the modified nucleoside O6-methylguanosine. Solid-phase oligoribonucleotide assembly was carried out by use of 2'-silyl-protected nucleoside phosphoramidites, a new O6-methylguanosine-containing synthon and a mild deprotection method. The O6-methylguanosine-modified oligonucleotides were used in the study of the role of conserved residues G5, G8 and G12 in hammerhead ribozyme cleavage. Hammerheads thus substituted at any of these positions showed an approximately 75-fold reduction in kcat whereas Km was unaffected. Hammerheads with modifications at G5 or G8 showed a significant reduction in magnesium binding affinity whereas modification at G12 had no effect. The results show that the three conserved G residues play crucial but different role sin hammerhead cleavage.

Base Sequence↗
PubMed1993

Human immunodeficiency virus type 1 transactivator protein, tat, stimulates transcriptional read-through of distal terminator sequences in vitro.

The human immunodeficiency virus type 1 transactivator protein, tat, specifically stimulates transcription from the viral long terminal repeat. We used cell-free transcription systems to test whether tat can stimulate transcriptional read-through of an artificial terminator sequence (e.g., a stable RNA stem-loop structure followed by a tract of nine uridine residues) placed downstream of the viral long terminal repeat. In the absence of tat, RNA polymerases are prematurely released from the template at the terminator sequence. Recombinant tat protein purified from Escherichia coli increased the synthesis of full-length transcripts approximately 25-fold and decreased the amount of transcripts ending at the terminator sequence. The reaction is strictly dependent upon the presence of a functional transactivation-responsive region (TAR) sequence. Mutations in the tat binding site on TAR RNA and mutations in the TAR RNA loop block transactivation in vivo. Neither type of mutation is able to respond to tat in vitro. These results strongly suggest that after transcription through the TAR region, tat modifies the transcription complex to increase its elongation capacity.

Base Sequence↗
PubMed1993

High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region.

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

Base Sequence↗
PubMed1993

RNA recognition by the human immunodeficiency virus Tat and Rev proteins.

The human immunodeficiency virus (HIV-1) regulatory proteins, Tat and Rev, are important potential targets for the development of new drug therapies against HIV infection. Both proteins are highly specific RNA-binding proteins that recognize cis-acting regulatory elements in the viral mRNAs. These interactions are fascinating paradigms of a new principle of RNA recognition in which the protein makes contact with functional groups displayed in a distorted major groove of an RNA duplex.

Base Sequence↗
PubMed1993

HIV gene regulatory proteins tat and rev and their interactions with synthetic RNA.

Synthetic oligoribonucleotides have been prepared and annealed to form model RNA duplexes that mimic the high affinity RNA recognition sites for the HIV-1 tat and rev proteins. The contributions of individual functional groups on the model RNAs to the specificity of binding by their respective proteins were studied by use of oligoribonucleotides containing site-specifically modified nucleotides. Both tat and rev appear to recognise specifically a limited number of functional groups in the major groove of an RNA double helix distorted by virtue of unpaired or non-Watson-Crick paired nucleotides.

Base Sequence↗