Jonathan Karn

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.

Periodic features in the amino acid sequence of nematode myosin rod.

Properties of the amino acid sequence of the nematode myosin rod region, deduced from cloned DNA, are analysed. The rod sequence of 1117 residues contains a regular region of 1094 residues, which has features typical of an alpha-helical coiled coil, followed by a short non-helical tailpiece at the carboxyl end. The hydrophobic amino acids show the expected seven-residue pattern a, b, c, d, e, f, g, which is modulated by a longer repeat of 28-residue zones. In addition, there are four one-residue insertions, or skip residues, at the ends of zones, at positions 351, 548, 745 and 970. Myosin is considerably less hydrophobic than tropomyosin or alpha-keratin and the outer surface of the coiled coil is covered by clusters of positive and negatively charged amino acid side-chains. Molecular models suggest that the coiled coil is continuous throughout the rod, with an approximately uniform left-handed twist, except for a few turns of helix near each skip region, where the twist flattens out to accommodate the extra residue. Fourier transforms of the amino acid profiles show strong periodicities based on repeats of seven residues (7/2 and 7/3) and 28 residues (especially 28/3 and 28/9). The positive and negative charges each have strong 28/3-residue periodicities that are out of phase with one another. The negative charges also show a 196/9-residue modulation frequency, which may reflect the presence of a 196-residue structural unit in muscle, approximately 2 X 143 A long. The distribution of charged amino acids suggests that electrostatic forces are dominant in forming the thick filament structure. Models that allow regular patterns of interacting charges are restricted and the simplest types are discussed.

RNA recognition by the human immuno-deficiency 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.

Activation of Human Immunodeficiency Virus Transcription in T Cells Revisited: NF-κB p65 Stimulates Transcriptional Elongation

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is able to establish a persistent latent infection during which the integrated provirus remains transcriptionally silent. Viral transcription is stimulated by NF-κB, which is activated following the exposure of infected T cells to antigens or mitogens. Although it is commonly assumed that NF-κB stimulates transcriptional initiation alone, we have found using RNase protection assays that, in addition to stimulating initiation, it can also stimulate elongation from the HIV-1 long terminal repeat. When either Jurkat or CCRF/CEM cells were activated by the mitogens phorbol myristate acetate and phytohemagglutinin, elongation, as measured by the proportion of full-length transcripts, increased two- to fourfold, even in the absence of Tat. Transfection of T cells with plasmids carrying the different subunits of NF-κB demonstrated that the activation of transcriptional elongation is mediated specifically by the p65 subunit. It seems likely that initiation is activated because of NF-κBs ability to disrupt chromatin structures through the recruitment of histone acetyltransferases. To test whether p65 could stimulate elongation under conditions where it did not affect histone acetylation, cells were treated with the histone deacetylase inhibitor trichostatin A. Remarkably, addition of p65 to the trichostatin A-treated cell lines resulted in a dramatic increase in transcription elongation, reaching levels equivalent to those observed in the presence of Tat. We suggest that the activation of elongation by NF-κB p65 involves a distinct biochemical mechanism, probably the activation of carboxyl-terminal domain kinases at the promoter.

Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans.

We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism.

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.

A new selective phage cloning vector, λ2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI

Abstract An improved bacteriophage λ cloning vector, λ2001, has been constructed. The phage includes a 34-bp Polylinker oligonucleotide which introduces cleavage sites for Xba I, Sst I, Xho I, Eco RI, Hind III and Bam HI, and can accommodate 10-kb to 23-kb fragments. Inserts that destroy the Bam HI or Xho I cloning sites may be recovered by excision at flanking sites in the Polylinker sequence. Insertion of foreign DNA into λ2001 generates phage with a Spi − phenotype. The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not. In the course of this work, the Polylinker sequence was also introduced into M13mp8. This produced a new vector, M13mp12, with cloning sites for Eco RI, Sma I, Xba I, Sst I, Xho I, Bam HI, and Hind III.

TRANSFER OF TAT AND RELEASE OF TAR RNA DURING THE ACTIVATION OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 TRANSCRIPTION ELONGATION COMPLEX

The HIV‐1 trans‐activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans‐activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3‐fold more full‐length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5′ end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3′ end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co‐factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co‐factors.

The genes sup-7 X and sup-5 III of C. elegans suppress amber nonsense mutations via altered transfer RNA

The sup-5 III and sup-7 X suppressors in C. elegans have previously been shown to have many genetic properties in common with tRNA nonsense suppressors of microorganisms. We report here the results of two lines of investigation into the molecular basis of these suppressors. In one, which sought to determine the nature of suppressible alleles, we demonstrate through DNA sequencing studies that a suppressible allele, unc-54(e 1300) I, of the myosin heavy chain gene contains a C leads to T substitution, which changes a glutamine codon to amber terminator at residue 1903. In the other approach, which sought to define the nature of the suppressing activity, we show through in vitro translation studies that tRNA fractions from the suppressor strains, but not wild-type, promote the specific readthrough of amber terminators of three different messenger RNAs. We conclude that sup-5 and sup-7 result in readthrough of amber terminators in vivo through an altered tRNA.

New Bacteriophage Lambda Vectors with Positive Selection for Cloned Inserts

Publisher Summary This chapter discusses new bacteriophage lambda vectors with positive selection for cloned inserts. Molecular cloning methods eliminated the necessity for physical fractionation of DNA and permitted, for the first time, the isolation of eukaryotic structural genes. Most bacteriophage vectors are substitution vectors that require internal filler fragments to be physically separated from the vector arms before insertions of foreign DNA. Genomic DNA is partially digested with restriction endonucleases to produce a population of DNA fragments from which molecules 15–20 kb long are purified by agarose gel electrophoresis. Other derivatives of 1059 that introduce defined amber mutations or alter the restriction enzyme sites on the vector have also been constructed. Successful and efficient cloning requires highly purified restriction enzymes and active DNA ligase.