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Biomedical subjects

J Karn

Publications and source records attributed to J Karn.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1992

Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.

The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure.

Bacteriophage T7↗
PubMed1991

Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs.

The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd less than 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-affinity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long filamentous ribonucleoprotein structures containing polymers of rev bound to flanking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.

Base Sequence↗
PubMed1991

RNA binding by the tat and rev proteins of HIV-1.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Control of human immunodeficiency virus replication by the tat, rev, nef and protease genes.

Immediately after infection, human immunodeficiency virus directs the synthesis of three regulatory proteins tat, rev and nef that together allow the synthesis of the structural proteins of the virus after a delay of several hours. Viral mRNA production is controlled by the tat gene, which appears to stimulate elongation by RNA polymerase II, and the rev gene, which allows the accumulation of unspliced or partially spliced mRNAs in the cytoplasm. The nef gene is dispensible for virus growth but may limit virus spread by downregulating the levels of cellular surface proteins such as the CD4 receptor. Virus maturation also depends critically on the protease gene which allows the orderly rearrangement of the viral core structures in newly budded virions as well as the vpu and vif genes which allow efficient production of mature envelope glycoprotein.

Chromosome Mapping↗
PubMed1990

HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region.

HIV-1 Rev protein, purified from E. coli, binds specifically to an RNA transcript containing the 223 nucleotide long Rev response element (RRE) sequence. Rev binds to RRE in vitro with an apparent dissociation constant of 1 to 3 nM as determined by filter binding, gel mobility shift assays, or an immunoprecipitation assay using a monoclonal antibody specific for the Rev C-terminus. Antisense RRE sequences are bound by Rev with a 20-fold lower affinity than wild-type RRE sequences. The Rev-RRE complex forms even in the presence of a 10,000-fold molar excess of 16S rRNA, whereas formation of the low affinity antisense RRE-Rev complex is efficiently blocked by addition of excess 16S rRNA. A approximately 33 nucleotide fragment is protected from ribonuclease T1 digestion by the binding of Rev to RRE RNA, suggesting that Rev binds with high affinity to only a restricted region of the RRE. This protected fragment is unable to rebind Rev protein but has been mapped to a 71 nucleotide long Rev binding domain sequence that overlaps the protected fragment.

Amino Acid Sequence↗
PubMed1990

HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure.

The HIV-1 trans-activator protein, tat, is an RNA binding protein with a high affinity for a U-rich bulge near the tip of the stem in the RNA stem-loop structure encoded by the trans-activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6- to 8-fold lower affinity than wild-type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem-loop structure reduced tat binding 10-fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.

Base Sequence↗