Subunit localizations of zinc(II) in DNA-dependent RNA polymerase from Escherichia coli B

Miller, Jeffrey A.; Serio, Gary F.; Howard, Robert A.; Bear, John L.; Evans, John E.; Kimball, A. P.

doi:10.1016/0005-2795(79)90056-4

Cited by 35 publications

(25 citation statements)

References 21 publications

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“…This ambiguity was eliminated by better data obtained later which indicated that the two ions were evenly distributed between /3 and 0'. A similar result was also reported by Miller et al (1979). The finding that only the intrinsic Zn ion in the /3 subunit was substituted by the denaturation-reconstitution method further confirms the stoichiometric distribution of Zn ions between /3 and p' and is consistent with our previous observation that the intrinsic Zn ion located in the p' subunit could not be removed from the enzyme by 7 M urea under similar denaturation conditions .…”

Section: Resultssupporting

confidence: 92%

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

Chatterji¹,

Wu²

1982

Biochemistry

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A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion. The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively. The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site. The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme. Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not. The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000). The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion. These observations suggest that the substituted metal was located at the initiation site of the enzyme. The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product. It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.

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Section: Resultssupporting

confidence: 92%

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

Chatterji¹,

Wu²

1982

Biochemistry

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“…One of us several years ago had located one Zn(II) at the β subunit which can be replaced with other divalent metals under suitable conditions [5]. AAS tentatively assigned the other Zn(II) at the β′subunit [24]. Later, sequence analysis carried out simultaneously by us and another group showed the presence of a putative Zn finger domain at the N‐terminal of the β′ subunit [25,26].…”

Section: Resultsmentioning

confidence: 99%

Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence‐based prediction and ⁶⁵Zn blotting

Sujatha

Chatterji

1999

FEBS Letters

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The availability of repeating`Cys' and/or`His' units in a particular order prompts the prediction of Zn(II) finger motifs in a protein. Escherichia coli RNA polymerase has two tightly bound Zn(II) per molecule of the enzyme as detected by atomic absorption spectroscopy. One Zn(II) was identified to be at the L L subunit, whereas the other putative Zn(II) binding site has recently been predicted to be at the N-terminal half of the L LP P subunit, from primary sequence analysis. We show here that the L LP P subunit has no ability to bind 65 Zn(II). On the other hand, the N-terminal domain of the K K subunit has strong Zn(II) binding ability with no obvious functional implications.z 1999 Federation of European Biochemical Societies.

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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%

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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Subunit localizations of zinc(II) in DNA-dependent RNA polymerase from Escherichia coli B

Cited by 35 publications

References 21 publications

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence‐based prediction and ⁶⁵Zn blotting

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

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Subunit localizations of zinc(II) in DNA-dependent RNA polymerase from Escherichia coli B

Cited by 35 publications

References 21 publications

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence‐based prediction and 65Zn blotting

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

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Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence‐based prediction and ⁶⁵Zn blotting