Nucleic acid‐binding regions of the second‐largest subunit of <i>Drosophila</i> RNA polymerase II identified by Southwestern blotting

Kontermann, Roland E.; Bautz, Ekkehard K. F.

doi:10.1016/0014-5793(94)00380-7

Cited by 4 publications

(5 citation statements)

References 30 publications

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“….. Weilbaecher et al, 1994), in yeast class II (rpbl-14, Scafe et al, 1990;sua8, Berroteran et al, 1994) or class III (rpcl6O-112, Dieci et al, 1995) RNA polymerases were represented on this diagram by calculating their corresponding position in the Algo gene from the center of conserved region d. Hatched regions in (B) and (D) were described to bind nucleic acid (Allison et al, 1985;Kontermann et al, 1993;Kontermann and Bautz, 1994).…”

Section: Resultsmentioning

confidence: 99%

“…If this interpretation were correct, the DNA would be expected to slide at the base of the stalk. Accordingly, the epitope recognized by the anti-AI35 antibody was mapped 22 residues away from conserved domain C which was shown to bind DNA ( Figure 8B, Allison et al, 1985;Kontermann and Bautz, 1994). Alternatively, the antibody may interfere with a conformational change of the enzyme involving the fingerlike stalk.…”

Section: Discussionmentioning

confidence: 99%

“…Cross-linking experiments indicate that this subunit also contacts the DNA template (Gundelfinger, 1983;Bartolomew et al, 1993). Non-specific DNA binding sites were revealed within domains C and D of Drosophila melanogaster RNA polymerase II by South-Western analysis (Kontermann and Bautz, 1994).…”

Section: Introductionmentioning

confidence: 99%

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Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

et al. 1996

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Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three‐dimensional model of the enzyme. Images of antibody‐labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit‐specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta‐like subunit is located at the base of the finger‐like domain, whereas a sequence between conserved regions C and D of the beta'‐like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha‐like subunit AC40 and subunit AC19 which were found co‐localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

et al. 1996

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“…on May 11, 2018 by guest http://mcb.asm.org/ largest subunit of yeast RNA polymerase III thought to be associated with RNA product binding (22,25,34) are able to compensate for the SUP4-CTC2 defect, completely restoring in vivo suppression to SUP4 levels (50).…”

Section: Discussionmentioning

confidence: 99%

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Zecherle

Whelen

Hall

1996

Molecular and Cellular Biology

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We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the ؉1 and ؉2 nucleotides of pre-tRNA Tyr , had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide ؉1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5 nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the ؉1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5 ends of the nascent transcripts of many genes act to stabilize the early ternary complex.The faithful and efficient transcription of DNA into RNA is a primary regulatory point in gene expression. The relative rates at which different genes are transcribed by the same RNA polymerase are strongly influenced by sequences surrounding the transcription start site. Previous studies of these contextual effects have emphasized sequence differences in the formation of the initiation complex and in the selection of the initiation site. RNA polymerases, both bacterial and eukaryotic, preferentially initiate transcription with a purine residue (4,5,8,14,18,29,30). In contrast, pyrimidine-specific transcription start sites are far less frequent as judged by 5Ј-end determination of RNAs synthesized both in vivo and in vitro (5, 18); only 7% of 88 Escherichia coli promoters surveyed by Hawley and McClure (14) directed transcription start sites with either UTP or CTP, with pyrimidine-specific initiation occurring only in the absence of purines within a defined window (36). The specific binding of purines at the i site of E. coli RNA polymerase, both in the presence and in the absence of template DNA, suggests that the interaction of purine ribonucleotides with RNA polymerase is important for productive transcription initiation (1, 47-49). However, the precise role in this process of nucleic acid sequences at and immediately following position ϩ1 remains unclear.As a system for studying small or large quantitative effects upon the level of transcription of a gene, RNA polymerase III transcription of the SUP4 gene ...

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“…DNA blotting, ultraviolet cross-linking, and antibody inhibition studies indicated the involvement of subunit 1, the homologue of the E. coli RNA polymerase ␤Ј subunit, in DNA binding (1)(2)(3)(4)(5), whereas subunit 2, the homologue of the E. coli ␤ subunit, has binding sites for nucleotide substrates (6 -9) and DNA (1,10,11). S. cerevisiae pol II subunit 3 (RPB3) and the corresponding subunits from other organisms have a limited sequence similarity to the E. coli ␣ subunit.…”

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confidence: 99%

RNA Polymerase II Subunits 2, 3, and 11 Form a Core Subassembly with DNA Binding Activity

Kimura¹,

Ishiguro²,

Ishihama³

1997

Journal of Biological Chemistry

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RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe consists of 10 species of subunit polypeptide. We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni 2؉ affinity chromatography. After stepwise dissociation of the Rpb1-and Rpb3-tagged RNA polymerases fixed on Ni 2؉ -resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained. Since the complex consisting of Rpb2, Rpb3, and Rpb11 cannot be dissociated even after treatment with 6 M urea buffer, we propose that this complex represents a core subassembly of the RNA polymerase II, analogous to the ␣ 2 ␤ complex in the assembly of Escherichia coli RNA polymerase. Both the Rpb2-Rpb3-Rpb11 complex and the free Rpb1 protein showed DNA binding activity, although the affinity was weaker compared with the intact RNA polymerase.Eukaryotic nuclear RNA polymerases are all multisubunit enzymes consisting of some 10 species of polypeptide. Among three types of nuclear RNA polymerase, RNA polymerase II (pol II) 1 is involved in the synthesis of mRNA and thus plays a central role in gene transcription. The genes coding for all the putative subunits have been cloned and sequenced for three organisms, human, the budding yeast Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe; but for all these enzymes, little is known about the function(s) of each polypeptide.DNA blotting, ultraviolet cross-linking, and antibody inhibition studies indicated the involvement of subunit 1, the homologue of the E. coli RNA polymerase ␤Ј subunit, in DNA binding (1-5), whereas subunit 2, the homologue of the E. coli ␤ subunit, has binding sites for nucleotide substrates (6 -9) and DNA (1, 10, 11). S. cerevisiae pol II subunit 3 (RPB3) and the corresponding subunits from other organisms have a limited sequence similarity to the E. coli ␣ subunit. As in the case of the E. coli ␣ subunit, one enzyme protomer probably contains two molecules of RPB3, and this subunit forms a complex with RPB2 in an early step of the enzyme assembly in vivo (12, 13).The ␣-like sequence also exists in S. cerevisiae RPB11 and the corresponding subunits in other eukaryotes (14 -16). The RPB3 and RPB11 homologues of Arabidopsis thaliana pol II associate in vitro with each other (15). Molecular interaction between S. cerevisiae AC40 and AC19, which are RPB3 and RPB11 homologues present in both RNA polymerases I and III, was also suggested by genetic analysis (17).pol II purified from S. pombe contains 10 species of subunit (18), named Rpb1 to Rpb12 after the S. cerevisiae subunit nomenclature (19), lacking two subunits corresponding to RPB4 and RPB9 of S. cerevisiae pol II. The S. pombe genes coding for all 10 subunits have been cloned and sequenced (16, 20 -25). In the case of S. cerevisiae pol II, RPB4 and RPB7 are readily dissociated from the enzyme and form a heterodi...

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Nucleic acid‐binding regions of the second‐largest subunit of Drosophila RNA polymerase II identified by Southwestern blotting

Cited by 4 publications

References 30 publications

Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

RNA Polymerase II Subunits 2, 3, and 11 Form a Core Subassembly with DNA Binding Activity

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