Subunit location of the intrinsic divalent metal ions in RNA polymerase from Escherichia coli

Wu, Cheng‐Wen; Wu, Felicia Y.-H.; Speckhard, David C.

doi:10.1021/bi00644a008

Cited by 63 publications

(46 citation statements)

References 33 publications

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“…As noted by Hudson et al (19) for spinach chloroplast 1', there are four cysteine residues within 22 amino acids that could possibly form a zinc-binding pocket. RNA polymerase is a zinc-containing enzyme, and the 13' subunit of E. coli RNA polymerase contains at least one of the two Zn(II)-binding sites (29,46). The E. coli p1' subunit is also thought to be involved in binding the DNA template (13).…”

Section: Resultsmentioning

confidence: 99%

“…However, it has been proposed that sequence differences among zinc fingers may reflect variations on a common structural theme, allowing a diversity of functions and an alteration or relaxation of DNA sequence specificity (12,20). Alternatively, it is possible that the putative zinc-binding region functions catalytically in transcription or plays a structural role in maintaining the proper subunit arrangement of the enzyme (21,46).…”

Section: Resultsmentioning

confidence: 99%

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Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120

Bergsland

Haselkorn

1991

J Bacteriol

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RNA polymerases of cyanobacteria contain a novel core subunit, y, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including y, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoCl, rpoC2 and encode the 1, y, and B' subunits, respectively. RNA polymerase in prokaryotes generally consists of a catalytic core of four subunits (W313'a2) and a dissociable sigma factor, which confers promoter specificity (7,16,44). The basic structure of the enzyme was believed to be the same in all eubacteria until the RNA polymerase of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified (37). The cyanobacterial core RNA polymerase was found to contain, in addition to a ,B, a ,3', and two a's, a novel core subunit of 70 kDa designated y, which is absent from the RNA polymerases of other eubacteria. Western immunoblotting with antiserum to -y has shown that a serologically related -y protein is present in over 30 different cyanobacteria, representing the five major taxonomic subgroups (4a, 36). Thus, the -y subunit is a common feature of cyanobacterial RNA polymerases.Anti--y serum cross-reacts with the Escherichia coli ' subunit protein, suggesting homology between these subunits (36). A region of DNA homologous to part of the E. coli gene encoding 3' (rpoC) has been isolated from another cyanobacterium, Nostoc commune UTEX 584 (47). Sequencing of this region showed that homology to E. coli rpoC is split between two linked genes, rpoCl and rpoC2, in the Nostoc DNA (47). The DNAs of several plant chloroplasts have likewise been found to contain regions of sequence homology with E. coli rpoC (19,20,30,38,39). In chloroplasts, the blocks of homology are also distributed between two genes, rpoCl and rpoC2. These genes are linked and are thought to encode the P' and ,B" subunits of chloroplast RNA polymerase, respectively.The exact subunit composition of chloroplast RNA polymerase is unclear. Spinach chloroplast RNA polymerase was found to contain seven prominent polypeptides, some of which were serologically related to subunits of the E. coli RNA polymerase (26). DNA sequences potentially encoding protein equivalents to the E. coli a and ,B subunits (rpoA and * Corresponding author. rpoB) have been found in chloroplast genomes, in addition to the rpoC homologs (19,20,30,39,40). In a few cases, these genes have been shown to be expressed in chloroplasts. The protein product of the rpoA gene has been identified in maize and pea chloroplasts (33,34 Isolation ofAnabaena sp. strain PCC 7120 RNA polymerase genes. The 2.8-and 2.3-kb EcoRI fragments containing parts of the E. coli rpoB and rpoC genes, respectively, were isolated from pGB218 (2), which was a gift from C. Squires. The fragments were labeled with [a-32P]dCTP (3,000 Ci/ mmol; New England Nuclear) by the random priming method (11) and used to probe a Southern blot of Anabaena sp. strain PCC 7120 chromosomal DNA digested ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120

Bergsland

Haselkorn

1991

J Bacteriol

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“…E. coli RNAP contains two zinc ions per molecule of holoenzyme (Scrutton et al 1971). Experiments involving metal substitution and oxidative immobilization indicated that one zinc ion is associated with ␤Ј, whereas the other may be associated with either ␤Ј or ␤ (Wu et al 1977). However, similar experiments with the highly homologous enzyme from Bacillus subtilis revealed that both zinc ions are bound to ␤Ј (Halling et al 1977).…”

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confidence: 99%

A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly

Markov¹,

Naryshkina²,

Mustaev³

et al. 1999

Genes & Development

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All multisubunit DNA-dependent RNA polymerases (RNAP) are zinc metalloenzymes, and at least two zinc atoms are present per enzyme molecule. RNAP residues involved in zinc binding and the functional role of zinc ions in the transcription mechanism or RNAP structure are unknown. Here, we locate four cysteine residues in the Escherichia coli RNAP largest subunit, ␤, that coordinate one of the two zinc ions tightly associated with the enzyme. In the absence of zinc, or when zinc binding is prevented by mutation, the in vitro-assembled RNAP retains the proper subunit stoichiometry but is not functional. We demonstrate that zinc acts as a molecular chaperone, converting denatured ␤ into a compact conformation that productively associates with other RNAP subunits. The ␤ residues coordinating zinc are conserved throughout eubacteria and chloroplasts, but are absent from homologs from eukaryotes and archaea. Thus, the involvement of zinc in the RNAP assembly may be a unique feature of eubacterial-type enzymes.

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“…The subunit has been implicated in binding substrates and products and contains at least a portion of the catalytic site for RNA synthesis. and 1' each bind one atom of Zn2+ (12,22). Sequence conservation among procaryotic and eucaryotic RNA polymerases suggests that the two large subunits of eucaryotic RNA polymerases bind to one another and have functions similar to their procaryotic counterparts.…”

mentioning

confidence: 99%

Genetic exploration of interactive domains in RNA polymerase II subunits.

1990

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The two large subunits of RNA polymerase H, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpbl-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpbl-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpbl-l suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.Yeast RNA polymerase II is composed of 10 polypeptide subunits whose molecular masses range from 190 to 10 kilodaltons (20). The genes that encode RPB1 and RPB2, the two largest subunits, have been cloned and sequenced (2, 21). RPB1 and RPB2 show extensive sequence similarity to the Escherichia coli RNA polymerase subunits P' and P, respectively. The areas of most extensive sequence similarity between the procaryotic and eucaryotic subunits have been termed homology regions (9). RPB1 has eight regions of homology, regions A through H (2), and RPB2 has nine, regions A through I (21). The presence of these regions of conserved amino acids has led to the suggestion that they define separate protein domains, and it is possible that each of these domains has a specific function that is conserved among RNA polymerases (18).Multiple functions have been attributed to the two large subunits of procaryotic and eucaryotic RNA polymerases. The E. coli RNA polymerase has been most extensively studied in this regard (4,23 Suppression of conditional mutations is a genetic means of identifying genes whose protein products interact with the product of a mutated gene (6). Suppression genetics has been used to reveal protein-protein interactions in the large multisubunit complexes of bacterial ribosomes (14) and eucaryotic flagella (10). It has also led to the isolation of new conditional mutations in P22 bacteriophage genes that encode structural components of the phage and permitted an analysis of the path of assembly of these components (8). The a-tubulin gene of Aspergillus nidulans was isolated by suppression of a conditional ,B-tubulin mutation (13). An actin-associating protein was isolated by suppression of a conditional mutation in actin (1). Suppressors of a Salmonel...

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Subunit location of the intrinsic divalent metal ions in RNA polymerase from Escherichia coli

Cited by 63 publications

References 33 publications

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120

A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly

Genetic exploration of interactive domains in RNA polymerase II subunits.

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