Yeast RNA‐polymerase B: A zinc protein

Lattke, Herbert; Weser, Ulrich

doi:10.1016/0014-5793(76)80131-7

Cited by 44 publications

(20 citation statements)

References 21 publications

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“…Nonchelating analogues of 1 ,1O-phenanthroline, such as 1,7-~henanthroline, do not inhibit the enzymes, which is consistent with the interpretation that the inhibition observed with 1,lO-phenanthroline results from chelation instead of nonspecific interactions. In addition to the polymerase reaction, chelating agents also inhibit the reverse reaction (i.e., pyrophosphorolysis) as well as pyrophosphate exchange among substrates (Seal and Loeb, 1976;Lattke and Weser, 1976). However, 1,lO-phenanthroline does not inhibit the 3' to 5' exonuclease activity of DNA polymerase I, which is thought to be involved in proofreading the newly synthesized polynucleotide chain (Que et al, 1979).…”

Section: Properties Of the Metal Centersmentioning

confidence: 96%

The Metallobiochemistry of Zinc Enzymes

Vallée

Galdes

1984

Advances in Enzymology - And Related Areas of Molecular Biology

175

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The honour and opportunity of being invited to address you on the occasion of Professor Houtman's retirement is very much appreciated. However, having met him yesterday, I am convinced that I am not here to help celebrate his retirement as much as to initiate his new career.Just like careers, the role of metals in biology can be viewed from multiple points of view. In the past, metals have variously been thought to be beneficial, toxic or to serve no purpose at all in biology. It is not very long ago that documentation for the validity of all th ree perceptions seemed to coexist, and that they had equally vigorous advocates; but by now the hypothesis that they serve no biological purpose has faded into oblivion. This dilemma owed much to the fact that many metals are present in 'trace' amounts. I want to address myself today to the general principles underlying the evolution of metallobiochemistry and of its subdisciplines which did not exist a generation ago and give perspective to metals as toxic, environmental agents, a subject which has been of special interest to Prof. Houtman.The word 'trace' implies something that can be measured qualitatively but not quantitatively, and until about a generation ago that was true for many metals in biology. Therefore it was a nearly mystical subject explored of ten by individuals who had ideas about the problem but who performed few experiments to verify or reject them. This has changed. Measurement of very low concentrations of metals in biology has become quite routine. Technical problems have been eliminated to the point where the term 'trace' has become meaningless. That is fortunate, indeed, because to many 'trace' also meant 'unimportant' or 'insignificant' .Attention to the detection limits of analytical methods for metals has become almost irrelevant now. Yet, the fact that the II-B metals and those of the first transition series all occur in biological matter in similarly low concentrations led to the tacit inference that all of them would be found to perform similar functions. Such an emphasis on similarities of concentrations completely ignored the chemical properties of metals which are unique for each. Not too surprisingly, their biological roles have turned out to be as characteristic as their chemistries, enhanced by co-ordination of metals with 132 12 (1988) DELFT PROGRESS REPORT

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Section: Properties Of the Metal Centersmentioning

confidence: 96%

The Metallobiochemistry of Zinc Enzymes

Vallée

Galdes

1984

Advances in Enzymology - And Related Areas of Molecular Biology

175

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“…ometry of zinc in eukaryotic RNA polymerases has been estimated to be 2.4 g-atoms of zinc per mol of enzyme for yeast RNA polymerase A (2), 1 g-atom in RNA polymerase B (23), and 4.3 g-atoms in RNA polymerase C (52). The former values are underestimates, since not only the two largest subunits but also some small subunits of yeast RNA polymerases A, B, and C were recently shown to bind zinc in vitro (48).…”

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confidence: 99%

Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.

Werner¹,

Denmat²,

Treich³

et al. 1992

Mol. Cell. Biol.

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The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.RNA polymerases belong to two different classes. One is represented by the RNA polymerases of SP6, T-uneven phages, and mitochondria that have polymerizing activity associated with a single polypeptide. The second class is made up of multisubunit enzymes found in eubacterial, archaebacterial, and eukaryotic cells (see references 41 and 47 for reviews). The two largest subunits of these enzymes are well conserved in all cellular enzymes, as indicated initially by immunological studies (reviewed in reference 40) and by subsequent comparison of the amino acid sequences deduced from the DNA sequences of the cloned genes (see references 41 and 47 for reviews). Comparison of the largest subunits of eukaryotic RNA polymerases A, B, and C and of the archaebacterial enzymes disclosed eight regions of homology, which have been termed domain a to domain h (20,41,47). Similarly, the second-largest subunit can be divided into a succession of conserved domains (41, 46). Most but not all of these domains are also present in the cognate eubacterial large subunits P' and ,B. Since the two largest subunits account for around 70% of the molecular mass of RNA polymerases, it is reasonable to propose that conserved domains participate in the catalytic functions of these enzymes. Some biochemical and genetic evidence supports this view. For instance, mutations affecting the nucleotidebinding site (34) and transcription termination (18,22) are located within conserved domains of the P-like subunit.Little is known of the structural or catalytic roles played by the largest subunit of RNA polymerases. Several genetic analyses have shown that RNA polymerase conditional mutations in the largest subunit tend to map in the conserved domains (14,38,53). Moreover, the fact that all a-amanitinresistant mutations affect the largest subunit of the B enzyme (see reference 37 for a review) suggests that the largest subunit is implicated in chain elongation. In the mouse, a-amanitin resistance results from an amino acid change in the strongly conserved domain f (3).The two large subunits are also involved in zinc binding.

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“…Adult host animals which developed ovaries (5). RNA polymerase II is known to bind zinc (24,26), so it is reasonable to speculate that RpII15 may sequester at least some of that zinc. The largest subunit of all three forms of eukaryotic RNA polymerase also contains a potential Znbinding domain (2,20,28).…”

Section: Materuils and Methodsmentioning

confidence: 99%

The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster.

Harrison¹,

Mortin²,

Corcés³

1992

Mol. Cell. Biol.

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A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster. Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II. The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species. Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains. The complex multimeric nature of RNA polymerases has been recognized for many years. RNA polymerases in eukaryotes consist of 9 to 14 subunits (for reviews, see references 43, 45, and 53). Some of these subunits are similar or identical in all three forms of polymerase in all species, while others are form and species specific. The largest subunits of RNA polymerase forms I, II, and III of the yeast Saccharomyces cerevisiae are sufficiently similar to allow detection of all three proteins by using antibodies generated to any one of the peptides (18). Furthermore, the fifth-, sixth-, and eighth-largest subunits (RPB5, RPB6, and RPB8) of RNA polymerase II are not just similar to those of the other polymerases but are shared between all three forms in yeast cells (51). This sequence conservation is not restricted to S. cerevisiae. The largest subunit of RNA polymerase II in yeast cells has regions with a high degree of interspecies homology, including similarity to Drosophila, mammalian, and even prokaryotic RNA polymerases (2,3,7,11,15 MATERUILS AND METHODSIsolation and maintenance of Drosophila strains. Fly stocks were maintained at 22.5°C and 65% relative humidity. The RpII15Z23 allele was generated as a lethal mutation in combination with Df(3R) red"52. Males homozygous for red, ebony were fed 0.024 M ethyl methanesulfonate (25) and mated to doubly balanced TM6B/TM3 females. Progeny were mated to Df(3R) red"52/TM6B flies. Mutations which resulted in no Df(3R) red 52/red, ebony flies thus delineated lethal complementation groups within the red"52 deficiency.Isolation and enzymology of nucleic acids. Isolation of plasmid DNA, screening of lambda libraries, and DNA labeling and enzymology were carried out by standard procedures (27). Genomic DNA from Drosophila adults was prepared as described by Parkhurst et al. (35). Total RNA was isolated by homogenization in 10 mM Tris hydrochloride (pH 7.4)-0.1 M NaCl-1 mM EDTA-0.5% sodium dodecyl sulfate followed by phenol extraction and ethanol precipitation. Poly(A)+ RNA was selected by chromatography on oligo(dT)-cellulose (4). Southern and Northern (RNA) analyses were done as described by Parkhurst et al. (35).

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Yeast RNA‐polymerase B: A zinc protein

Cited by 44 publications

References 21 publications

The Metallobiochemistry of Zinc Enzymes

The Metallobiochemistry of Zinc Enzymes

Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.

The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster.

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