Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

https://science.sciencemag.org/content/sci/277/5328/955.full.pdf

Telomerase catalytic subunit homologs from fission yeast and human

A combinatorial disulfide cross-linking strategy was used to prepare a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase with a DNA template:primer and a deoxynucleoside triphosphate (dNTP), and the crystal structure of the complex was determined at a resolution of 3.2 angstroms. The presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the molecular basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chemical structure of the drug.

https://science.sciencemag.org/content/sci/282/5394/1669.full.pdf

Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

https://science.sciencemag.org/content/sci/276/5312/561.full.pdf

Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase

Zinc enzymology is, compared to some other current areas of metallobiochemistry, a maturing field, but in addition to further developments of structure-function relationships it has also provided a number of surprising new results and ideas in the last few years. In fact, the number of studies makes it impossible to provide a comprehensive review of the recent literature on zinc enzymology here, and the authors therefore focus on those zinc enzymes for which structure-function relationships are possible on the basis of structural and biochemical data. This means that, with a few exceptions, only zinc enzymes for which NMR or crystal structures are available are included here. Another seemingly simple, yet experimentally sometimes complex issue concerns the choice of which metalloenzyme is a zinc enzyme. Since there is in principle no difference in chemical catalysis by low-affinity compared to high-affinity metal sites, some of these enzymes are also included in this article, especially if they are or have been discussed as zinc enzymes, or are active with zinc. 552 refs.

Recent Advances in Zinc Enzymology

Human immunodeficiency virus type 1 is a complex retrovirus encoding 15 distinct proteins. Substantial progress has been made toward understanding the function of each protein, and three-dimensional structures of many components, including portions of the RNA genome, have been determined. This review describes the function of each component in the context of the viral life cycle: the Gag and Env structural proteins MA (matrix), CA (capsid), NC (nucleocapsid), p6, SU (surface), and TM (transmembrane); the Pol enzymes PR (protease), RT (reverse transcriptase), and IN (integrase); the gene regulatory proteins Tat and Rev; and the accessory proteins Nef, Vif, Vpr, and Vpu. The review highlights recent biochemical and structural studies that help clarify the mechanisms of viral assembly, infection, and replication.

http://www3.uah.es/farmamol/Public/AnnReviews/PDF/Biochemistry/HIV1.pdf

HIV-1: fifteen proteins and an RNA.

The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nucleophilic attack on the alpha-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.

https://www.pnas.org/content/pnas/90/13/6320.full.pdf

P Clark

Papers

Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA