Two conformations of RNA polymerase II revealed by electron crystallography

Asturias, Francisco J.; Meredith, Gavin; Poglitsch, Claudia L.; Kornberg, Roger D.

doi:10.1006/jmbi.1997.1273

Cited by 52 publications

(44 citation statements)

References 22 publications

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“…The very high conservation of sequence, structure, and function between E. coli and Taq (Fig. 1), and the fact that related conformational changes of a similar scale have been observed in different crystal forms and different functional complexes of yeast RNAP II (24,(27)(28)(29)(30), leads to the conclusion that the observed conformational change reflects the normal flexibility of the RNAP rather than differences between E. coli and Taq RNAPs or crystallization artifacts.…”

Section: Discussionmentioning

confidence: 96%

Conformational flexibility of bacterial RNA polymerase

Darst

Opalka

Chacón

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

109

113

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The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the highresolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA͞RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.

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Section: Discussionmentioning

confidence: 96%

Conformational flexibility of bacterial RNA polymerase

Darst

Opalka

Chacón

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

109

113

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“…The RNA polymerase II from animals, plants, and the fission yeast might be fixed at the form which is capable of accepting subunit 4. The S. cerevisiae RNA polymerase II, saturated with RPB4, from cells under stress conditions forms high-quality two-dimensional crystals (1,9). The S. pombe RNA polymerase II, which is tightly associated with Rpb4 even under normal growth conditions, could be a good tool for analysis of threedimensional structure of the RNA polymerase II.…”

Section: Discussionmentioning

confidence: 99%

The Rpb4 Subunit of Fission Yeast Schizosaccharomyces pombe RNA Polymerase II Is Essential for Cell Viability and Similar in Structure to the Corresponding Subunits of Higher Eukaryotes

Sakurai

Mitsuzawa

Kimura

et al. 1999

Molecular and Cellular Biology

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Both the gene and the cDNA encoding the Rpb4 subunit of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. The cDNA sequence indicates that Rpb4 consists of 135 amino acid residues with a molecular weight of 15,362. As in the case of the corresponding subunits from higher eukaryotes such as humans and the plant Arabidopsis thaliana, Rpb4 is smaller than RPB4 from the budding yeast Saccharomyces cerevisiae and lacks several segments, which are present in the S. cerevisiae RPB4 subunit, including the highly charged sequence in the central portion. The RPB4 subunit of S. cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S. pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase II containing RPB4 in exponentially growing cells of S. cerevisiae is about 20%, but S. pombe RNA polymerase II contains the stoichiometric amount of Rpb4 even at the exponential growth phase. In contrast to the RPB4 homologues from higher eukaryotes, however, S. pombe Rpb4 formed stable hybrid heterodimers with S. cerevisiae RPB7, suggesting that S. pombe Rpb4 is similar, in its structure and essential role in cell viability, to the corresponding subunits from higher eukaryotes. However, S. pombe Rpb4 is closer in certain molecular functions to S. cerevisiae RPB4 than the eukaryotic RPB4 homologues.RNA polymerase II in eukaryotes is composed of more than 10 different polypeptides (for example, see reference 29). The genes coding for all 12 putative subunits of RNA polymerase II have been isolated from the budding yeast Saccharomyces cerevisiae (reviewed in references 26 and 27) and humans (10). Sometime ago we reported that the purified RNA polymerase II from the fission yeast Schizosaccharomyces pombe contained at least 10 polypeptides, devoid of the components corresponding to RPB4 and RPB9 of S. cerevisiae (21, 24; for a recent review, see reference 8). Later we cloned the gene and the cDNA for Rpb9 by PCR using the sequence knowledge of subunit 9 from other organisms (23). By Western blot analysis with antibodies against the Rpb9 protein expressed in Escherichia coli, we found that the purified S. pombe RNA polymerase II does indeed contain Rpb9, which had not been detected in the gel pattern because of its comigration with Rpb8 and Rpb11 (23).Recently, the genes coding for subunit 4 were cloned from humans (10) and the plant Arabidopsis thaliana (15). Human cDNA for RPB4 was cloned by two-hybrid screening of cDNA coding for a protein which interacts with human RPB7 (hRPB7) (10), because S. cerevisiae RPB4 forms a binary complex with RPB7 (6, 11). On the other hand, the gene for the A. thaliana RPB15.9 (AtRPB15.9) subunit, which is a homologue of S. cerevisiae RPB4, was cloned by cross-hybridization using the homologous expressed sequence tag (EST) clone of oilseed rape (Brassica napus) as the probe (15). Both hRPB4 and AtRPB15.9 are smaller than S. cerevisiae RPB4, lacking a segment cor...

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“…This defect can be reversed in vitro by the addition of purified RNAP II (7,19) or in vivo upon high copy expression of the RPB7 gene in ⌬RPB4 cells (10). Structural studies of RNAP II purified from ⌬RPB4 cells compared with the wild type enzyme also reveal that the portion of the enzyme that clamps DNA exists in a more open, less stable conformation (8,9). Based on cumulative structural data (placing RPB4 and RPB7 downstream of the catalytic site in the center of the 25-Å cleft of the enzyme; Ref.…”

Section: Rnap II Loss Of Function Also Affects Rnap I But Not Rnap Imentioning

confidence: 99%

“…Therefore, the most homogenous preparations of RNAP II are obtained from ⌬RPB4 cells and used as a source of the enzyme for most general structural studies. Comparison of a lower resolution structure of the entire 12-subunit enzyme to the high resolution structure of the enzyme obtained from ⌬RPB4 cells revealed differences in conformation; the wild type enzyme favors the closed conformation, and the mutant enzyme favors the open conformation (8,9). RNAP II lacking the RPB4⅐RPB7 subcomplex forms a stable preinitiation complex with general transcription factors, and consequently, this subcomplex is required for a step following template commitment (7).…”

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confidence: 99%

Deletion of the RNA Polymerase Subunit RPB4 Acts as a Global, Not Stress-specific, Shut-off Switch for RNA Polymerase II Transcription at High Temperatures

Miyao

Barnett

Woychik

2001

Journal of Biological Chemistry

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We used whole genome expression analysis to investigate the changes in the mRNA profile in cells lacking the Saccharomyces cerevisiae RNA polymerase II subunit RPB4 (⌬RPB4). Our results indicated that an essentially complete shutdown of transcription occurs upon temperature shift of this conditionally lethal mutant; 98% of mRNA transcript levels decrease at least 2-fold, 96% at least 4-fold. This data was supported by in vivo experiments that revealed a rapid and greater than 5-fold decline in steady state poly(A) RNA levels after the temperature shift. Expression of several individual genes, measured by Northern analysis, was also consistent with the whole genome expression profile. Finally we demonstrated that the loss of RNA polymerase II activity causes secondary effects on RNA polymerase I, but not RNA polymerase III, transcription. The transcription phenotype of the ⌬RPB4 mutant closely mirrors that of the temperature-sensitive rpb1-1 mutant frequently implemented as a tool to inactivate the RNA polymerase II in vivo. Therefore, the ⌬RPB4 mutant can be used to easily design strains that enable the study of distinct post-transcriptional cellular processes in the absence of RNA polymerase II transcription. RNA polymerase (RNAP)1 II is a highly conserved 12-subunit enzyme that is a component of a large protein complex involved in the regulated synthesis of eukaryotic mRNA (1, 2). Of the 12 yeast Saccharomyces cerevisiae RNAP II subunits, designated RPB1-RPB12, five have counterparts in bacterial RNAP. RPB1 and RPB2 are orthologs of the ␤Ј and ␤ subunits, respectively. RPB3 and RPB11 are structurally and functionally related to the bacterial ␣ subunit pair (3, 4), and RPB6 is the ortholog of the subunit (5).In addition to the functional parallels that exist between bacterial and eukaryotic RNAP II subunits, there is extensive functional similarity between subunits comprising the three classes of RNAP. All but one of the RNAP II subunits, RPB4, have some functional relationship to a corresponding subunit in RNAP I and RNAP III. Five of the subunits are also identical in RNAP I, II, and III. Six other RNAP II subunits are related in sequence to subunits in either or both RNAP I and RNAP III. Therefore, the relatively small RPB4 subunit (221 amino acids, 25 kDa) has a function exclusive to RNAP II.RPB4 interacts with RPB7, another small (171 amino acid, 19 kDa) essential subunit (6). This subunit pair can dissociate from the enzyme upon biochemical purification and deletion of the RPB4 gene from yeast cells results in diminished association of the RPB7 subunit with the enzyme. The purified RPB4⅐RPB7 subcomplex binds both single-stranded DNA and single-stranded RNA in vitro (7). A predicted oligosaccharide/ oligonucleotide binding fold in RPB7 is crucial for both the nucleic acid binding and transcription activity of the subunit pair (7). However, the two functions are not absolutely linked since one RPB7 mutant causes a loss of transcription activity without affecting nucleic acid binding (7).RPB4 and RPB7 are pr...

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Two conformations of RNA polymerase II revealed by electron crystallography

Cited by 52 publications

References 22 publications

Conformational flexibility of bacterial RNA polymerase

Conformational flexibility of bacterial RNA polymerase

The Rpb4 Subunit of Fission Yeast Schizosaccharomyces pombe RNA Polymerase II Is Essential for Cell Viability and Similar in Structure to the Corresponding Subunits of Higher Eukaryotes

Deletion of the RNA Polymerase Subunit RPB4 Acts as a Global, Not Stress-specific, Shut-off Switch for RNA Polymerase II Transcription at High Temperatures

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