scholarly journals Quantitative conservation of chromatin-bound RNA polymerases I and II in mitosis. Implications for chromosome structure.

1979 ◽  
Vol 80 (2) ◽  
pp. 451-464 ◽  
Author(s):  
S I Matsui ◽  
H Weinfeld ◽  
A A Sandberg
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Negative Control ◽  
Rna Polymerases ◽  
Chromatin Interaction ◽  
Condensed State ◽  
Metaphase Chromosomes ◽  
A Genome ◽  
Per Se

RNA synthesis almost ceases in mitosis. It is ambiguous whether this temporal, negative control of RNA synthesis is solely because of the nature of chromosomes per se, (i.e., their condensed state), or to a physical loss of RNA polymerases along with other nuclear proteins which have been shown to pass into the cytoplasm in mitosis, or to their combined feature. Aside from such regulatory considerations, a question has also been raised as to whether RNA polymerases are constituents of metaphase chromosomes. To clarify these aspects of RNA polymerase-chromatin interaction in mitosis, the enzymes in chromosomes were quantitated and their levels compared to those in interphase nuclei and cells at various phases of the cell cycle. The results show that the amounts of form I, form II, and probably form III enzymes bound to a genome-equivalent of chromatin stay constant during the cell cycle. Thus, the mechanism for the negative control of RNA synthesis in mitosis appears to exist in the chromosomes per se, but not to be directly related to the RNA polymerase levels. This quantitative conservation of chromatin-bound RNA polymerases implies that they may persist as structural components of the chromosomes in mitosis.

scholarly journals Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes.

1997 ◽  
Vol 17 (10) ◽  
pp. 5791-5802 ◽  
Author(s):  
G G Parsons ◽  
C A Spencer
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Transcription Elongation ◽  
Control Cell ◽  
Specific Gene ◽  
Mitotic Chromosomes ◽  
Transcription Repression ◽  
Rna Polymerase Ii Transcription

Nuclear RNA synthesis is repressed during the mitotic phase of each cell cycle. Although total RNA synthesis remains low throughout mitosis, the degree of RNA polymerase II transcription repression on specific genes has not been examined. In addition, it is not known whether mitotic repression of RNA polymerase II transcription is due to polymerase pausing or ejection of transcription elongation complexes from mitotic chromosomes. In this study, we show that RNA polymerase II transcription is repressed in mammalian cells on a number of specific gene regions during mitosis. We also show that the majority of RNA polymerase II transcription elongation complexes are physically excluded from mitotic chromosomes between late prophase and late telophase. Despite generalized transcription repression and stripping of RNA polymerase II complexes from DNA, arrested RNA polymerase II ternary complexes appear to remain on some gene regions during mitosis. The cyclic repression of transcription and ejection of RNA polymerase II transcription elongation complexes may help regulate the transcriptional events that control cell cycle progression and differentiation.

NTP-driven translocation and regulation of downstream template opening by multi-subunit RNA polymerases

2005 ◽  
Vol 83 (4) ◽  
pp. 486-496 ◽  
Author(s):  
Zachary F Burton ◽  
Michael Feig ◽  
Xue Q Gong ◽  
Chunfen Zhang ◽  
Yuri A Nedialkov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Binding Sites ◽  
Rna Synthesis ◽  
Rna Polymerases ◽  
Phosphoryl Transfer ◽  
Elongation Complex ◽  
Detailed Model ◽  
Loop 2

Multi-subunit RNA polymerases bind nucleotide triphosphate (NTP) substrates in the pretranslocated state and carry the dNMP–NTP base pair into the active site for phosphoryl transfer. NTP-driven translocation requires that NTP substrates enter the main-enzyme channel before loading into the active site. Based on this model, a new view of fidelity and efficiency of RNA synthesis is proposed. The model predicts that, during processive elongation, NTP-driven translocation is coupled to a protein conformational change that allows pyrophosphate release: coupling the end of one bond-addition cycle to substrate loading and translocation for the next. We present a detailed model of the RNA polymerase II elongation complex based on 2 low-affinity NTP binding sites located in the main-enzyme channel. This model posits that NTP substrates, elongation factors, and the conserved Rpb2 subunit fork loop 2 cooperate to regulate opening of the downstream transcription bubble.Key words: RNA polymerase, NTP-driven translocation, transcriptional fidelity, transcriptional efficiency, α-amanitin.

scholarly journals Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

1998 ◽  
Vol 72 (8) ◽  
pp. 6546-6553 ◽  
Author(s):  
Julie A. Lemm ◽  
Anders Bergqvist ◽  
Carol M. Read ◽  
Charles M. Rice
Keyword(s):  
Rna Polymerase ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Functional Assay ◽  
Minus Strand ◽  
Virus Rna ◽  
A Genome ◽  
Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

Phosphorylation of RNA polymerases: specific association of protein kinase NIl with RNA polymerase I

1983 ◽  
Vol 302 (1108) ◽  
pp. 135-142 ◽  
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Polymerase Iii ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Liver Protein ◽  
Protein Kinase N ◽  
Polymerase I

The activities of the three DNA-dependent RNA polymerases from a rapidly growing rat tumour, Morris hepatoma 3924 A, and from rat liver were examined. The activity of RNA polymerase I was higher in the tumour than in the liver. The enhanced capacity for RNA synthesis was a result of a higher concentration of polymerase I in the tumour as well as of an activation of this enzyme vivo. The possibility that the high specific activity of the hepatoma polymerase I resulted from phosphorylation was investigated. Two major cyclic-AMP-independent nuclear casein kinases (NI and N il) were identified; the activity of protein kinase N il in the tumour was ten times that in liver. Protein kinase N il was capable of activating and phosphorylating RNA polymerase I in vitro . This kinase could also stimulate RNA polymerase II activity, although to a lesser extent than RNA polymerase I. RNA polymerase III was not affected by protein kinase NIL Protein kinase N il was tightly associated with polymerase I and was found even in purified preparations of the polymerase. Antibodies against both RNA polymerase I and protein kinase N il were present in sera of patients with certain rheumatic autoimmune diseases. These results imply that RNA polymerase I and protein kinase NIl are in close association in vivo as well as in vitro and that polymerase phosphorylation may regulate the rate of ribosomal RNA synthesis in the cell.

scholarly journals Role of Second-Largest RNA Polymerase I Subunit Zn-Binding Domain in Enzyme Assembly

2003 ◽  
Vol 2 (5) ◽  
pp. 1046-1052 ◽  
Author(s):  
Tatyana Naryshkina ◽  
Adrian Bruning ◽  
Olivier Gadal ◽  
Konstantin Severinov
Keyword(s):  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Cysteine Residues ◽  
Per Se ◽  
Essential Function ◽  
Polymerase I

ABSTRACT The second-largest subunits of eukaryal RNA polymerases are similar to the β subunits of prokaryal RNA polymerases throughout much of their lengths. The second-largest subunits from eukaryal RNA polymerases contain a four-cysteine Zn-binding domain at their C termini. The domain is also present in archaeal homologs but is absent from prokaryal homologs. Here, we investigated the role of the C-terminal Zn-binding domain of Rpa135, the second-largest subunit of yeast RNA polymerase I. Analysis of nonfunctional Rpa135 mutants indicated that the Zn-binding domain is required for recruitment of the largest subunit, Rpa190, into the RNA polymerase I complex. Curiously, the essential function of the Rpa135 Zn-binding domain is not related to Zn2+ binding per se, since replacement of only one of the four cysteine residues with alanine led to the loss of Rpa135 function. Even more strikingly, replacement of all four cysteines with alanines resulted in functional Rpa135.

scholarly journals Increased numbers of nucleoli in a genome-wide RNAi screen reveal proteins that link the cell cycle to RNA polymerase I transcription

2021 ◽  
Vol 32 (9) ◽  
pp. 956-973 ◽  
Author(s):  
Lisa M. Ogawa ◽  
Amber F. Buhagiar ◽  
Laura Abriola ◽  
Bryan A. Leland ◽  
Yulia V. Surovtseva ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Ribosome Biogenesis ◽  
Rna Polymerase I ◽  
Wide Field ◽  
Genome Wide ◽  
A Genome ◽  
Nucleolar Number ◽  
Polymerase I ◽  
Cycle Regulation

The nucleolus is a dynamic nuclear condensate and site of ribosome biogenesis. Using wide-field fluorescence microscopy, we screened for proteins that when depleted cause an increase in nucleolar number. Our results uncovered an unexpected subset of proteins that link the nucleolus, cell cycle regulation, and RNA polymerase I transcription.

Template activity of unfixed metaphase chromosomes

1976 ◽  
Vol 20 (1) ◽  
pp. 215-219
Author(s):  
B.B. Cohen ◽  
D.L. Deane
Keyword(s):  
Cell Membrane ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Mitotic Cycle ◽  
Metaphase Chromosomes ◽  
Template Activity ◽  
Transcription Process

Unfixed metaphase and non-metaphase cells were tested for their template activity with RNA polymerase. A device was used which disrupts the cell membrane by centrifugation, and which also ensures that the cells do not continue with their mitotic cycle during the transcription process. The template activity of acid/methanol fixed cells was also tested. None of the unfixed metaphase cells transcribed RNA whereas most of the non-metaphase cells did. In contrast, using fixed cells both classes of cells were transcribed equally well and to a much greater extent. It was concluded that metaphase chromosomes in vivo cannot act as templates for RNA synthesis.

scholarly journals High concentration of RNA polymerase I is responsible for the high rate of nucleolar transcription

1980 ◽  
Vol 188 (2) ◽  
pp. 381-385 ◽  
Author(s):  
F L Yu
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
High Rate ◽  
Unit Weight ◽  
High Concentration ◽  
Steady State Condition ◽  
Polymerase I ◽  
Nucleolar Rna

When isolated rat liver nuclei and nucleoli are compared for RNA synthesis in vitro, the rate of nucleolar RNA synthesis is found to be more than 10 times higher. In order to understand this high rate of nucleolar transcription, DNA from both nuclear and nucleolar fractions was isolated and compared for the ability to direct RNA synthesis with homologous RNA polymerases. No difference between these two templates is evident. On the other hand, when the total nuclear and nucleolar RNA polymerases are isolated and compared on a per-unit-weight-of-DNA basis, it becomes clear that the nucleolus has a 10-fold higher RNA polymerase concentration than the nucleus. This result suggests that RNA polymerase I concentration rather than the nucleolar DNA template efficiency is responsible for the observed high rate of nucleolar transcription under the normal steady-state condition.

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