RNAP subunits F/E (RPB4/7) are stably associated with archaeal RNA polymerase: using fluorescence anisotropy to monitor RNAP assembly in vitro

2009 ◽  
Vol 421 (3) ◽  
pp. 339-343 ◽  
Author(s):  
Dina Grohmann ◽  
Angela Hirtreiter ◽  
Finn Werner
Keyword(s):  
Rna Polymerase ◽  
Molecular Interactions ◽  
Fluorescence Anisotropy ◽  
Rna Polymerases ◽  
Dynamic Exchange ◽  
Transcription Cycle

Archaeal and eukaryotic RNAPs (DNA-dependent RNA polymerases) are complex multi-subunit enzymes. Two of the subunits, F and E, which together form the F/E complex, have been hypothesized to associate with RNAP in a reversible manner during the transcription cycle. We have characterized the molecular interactions between the F/E complex and the RNAP core. F/E binds to RNAP with submicromolar affinity and is not in a dynamic exchange with unbound F/E.

scholarly journals RNA polymerase bypass at sites of dihydrouracil: implications for transcriptional mutagenesis.

1995 ◽  
Vol 15 (12) ◽  
pp. 6729-6735 ◽  
Author(s):  
J Liu ◽  
W Zhou ◽  
P W Doetsch
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerases ◽  
Dna Damages ◽  
Mutant Proteins ◽  
Dna Lesion ◽  
Transcriptional Mutagenesis ◽  
Rapid Generation ◽  
Transition Mutation

Dihydrouracil (DHU) is a major base damage product formed from cytosine following exposure of DNA to ionizing radiation under anoxic conditions. To gain insight into the DNA lesion structural requirements for RNA polymerase arrest or bypass at various DNA damages located on the transcribed strand during elongation, DHU was placed onto promoter-containing DNA templates 20 nucleotides downstream from the transcription start site. In vitro, single-round transcription experiments carried out with SP6 and T7 RNA polymerases revealed that following a brief pause at the DHU site, both enzymes efficiently bypass this lesion with subsequent rapid generation of full-length runoff transcripts. Direct sequence analysis of these transcripts indicated that both RNA polymerases insert primarily adenine opposite to the DHU site, resulting in a G-to-A transition mutation in the lesion bypass product. Such bypass and insertion events at DHU sites (or other types of DNA damages), if they occur in vivo, have a number of important implications for both the repair of such lesions and the DNA damage-induced production of mutant proteins at the level of transcription (transcriptional mutagenesis).

scholarly journals Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

2010 ◽  
Vol 30 (21) ◽  
pp. 5180-5193 ◽  
Author(s):  
Alicia García ◽  
Emanuel Rosonina ◽  
James L. Manley ◽  
Olga Calvo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Terminal Domain ◽  
Mrna Metabolism ◽  
Genes Encoding ◽  
The One ◽  
Ctd Phosphorylation ◽  
Transcription Cycle

ABSTRACT The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

scholarly journals Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2359-2366 ◽  
Author(s):  
Ming Tan ◽  
Tamas Gaal ◽  
Richard L. Gourse ◽  
Joanne N. Engel
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Rich Region ◽  
E Coli ◽  
Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

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 The Length of Promoter Sequence Affects The De Novo Initiation By T7 RNA Polymerase in Vitro: New Insights Into The Evolution of Promoters For Single Subunit RNA Polymerases

2019 ◽  
Author(s):  
Ramesh Padmanabhan ◽  
Dennis Miller
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
De Novo ◽  
Promoter Sequence ◽  
Rna Polymerases ◽  
T7 Rna Polymerase ◽  
Promoter Sequences ◽  
Promoter Recognition ◽  
Single Subunit

1.1AbstractRNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate de novo transcription. Promoter recognition requires the presence of specific DNA binding domains in the polymerase. The structure and mechanistic aspects of transcription by the bacteriophage T7 RNA polymerase (T7 RNAP) are well characterized. This single subunit RNAP belongs to the family of RNAPs which also includes the T3, SP6 and mitochondrial RNAPs. High specificity for its promoter, the requirement of no additional transcription factors, and high fidelity of initiation from a specific site in the promoter makes it the polymerase of choice to study the mechanistic aspects of transcription. The structure and function of the catalytic domains of this family of polymerases are highly conserved suggesting a common mechanism underlying transcription. Although the two groups of single subunit RNAPs, mitochondrial and bacteriophage, have remarkable structural conservation, they recognize quite dissimilar promoters. Specifically, the bacteriophage promoters recognize a 23 nucleotide promoter extending from −17 to + 6 nucleotides relative to the site of transcription initiation, while the well characterized promoter recognized by the yeast mitochondrial RNAP is nine nucleotides in length extending from −8 to +1 relative to the site of transcription initiation. Promoters recognized by the bacteriophage RNAPs are also well characterized with distinct functional domains involved in promoter recognition and transcription initiation. Thorough mutational studies have been conducted by altering individual base-pairs within these domains. Here we describe experiments to determine whether the prototype bacteriophage RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. When a complete or almost complete (20 to 16 nucleotide) double stranded T7 RNAP promoter sequence is present, small RNA’s are produced through template-independent and promoter-dependent stuttering corresponding to abortive initiation, and this effect was lost with a scrambled promoter sequence. When partial double stranded promoter sequences (10 to 12 nucleotides) are supplied, template dependent de novo initiation of RNA occurs at a site different from the canonical +1-initiation site. The site of transcription initiation is determined by a recessed 3’ end based paired to the template strand of DNA rather than relative to the partial promoter sequence. Understanding the mechanism underlying this observation helps us to understand the role of the elements in the T7 promoter, and provides insights into the promoter evolution of the single-subunit RNAPs.

scholarly journals Role for the Ssu72 C-Terminal Domain Phosphatase in RNA Polymerase II Transcription Elongation

2006 ◽  
Vol 27 (3) ◽  
pp. 926-936 ◽  
Author(s):  
Mariela Reyes-Reyes ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Elongation Complex ◽  
Terminal Domain ◽  
Rnap Ii ◽  
Transcription Cycle

ABSTRACT The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y1S2P3T4S5P6S7) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

scholarly journals N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

2002 ◽  
Vol 184 (18) ◽  
pp. 4952-4961 ◽  
Author(s):  
S. H. Willis ◽  
K. M. Kazmierczak ◽  
R. H. Carter ◽  
L. B. Rothman-Denes
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Rna Polymerases ◽  
Transcriptional Analysis ◽  
Amino Acid Residues ◽  
Genes Encoding ◽  
Single Subunit

ABSTRACT Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

scholarly journals Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements.

2008 ◽  
Vol 55 (3) ◽  
pp. 537-547
Author(s):  
Piotr Bonarek ◽  
Sylwia Kedracka-Krok ◽  
Barbara Kepys ◽  
Zygmunt Wasylewski
Keyword(s):  
Rna Polymerase ◽  
Fluorescence Anisotropy ◽  
Multicomponent System ◽  
Binding Constants ◽  
Receptor Protein ◽  
Activator Protein ◽  
Apparent Binding Constant ◽  
Transcription Complexes ◽  
Promoter Interaction

The in vitro formation of transcription complexes with Escherichia coli RNA polymerase was monitored using fluorescence anisotropy measurements of labeled fragments of DNA. The multicomponent system consisted of holo or core RNA polymerase (RNAP) and lac or gal promoter fragments of DNA (in different configurations), in the presence or absence of CRP activator protein (wt or mutants) with its ligand, cAMP. Values of the apparent binding constants characterizing the system were obtained, as a result of all processes taking place in the system. The interaction of the promoters with core RNAP in the absence of CRP protein was characterized by apparent binding constants of 0.67 and 1.9 x 10(6) M(-1) for lac166 and gal178, respectively, and could be regarded as nonspecific. The presence of wt CRP enhanced the strength of the interaction of core RNAP with the promoter, and even in the case of gal promoter it made this interaction specific (apparent binding constant 2.93 x 10(7) M(-1)). Holo RNAP bound the promoters significantly more strongly than core RNAP did (apparent binding constants 1.46 and 40.14 x 10(6) M(-1) for lac166 and gal178, respectively), and the presence of CRP also enhanced the strength of these interactions. The mutation in activator region 1 of CRP did not cause any significant disturbances in the holo RNAP-lac promoter interaction, but mutation in activator region 2 of the activator protein substantially weakened the RNAP-gal promoter interaction.

scholarly journals Transcriptionally active RNA polymerases from Morris hepatomas and rat liver. Elucidation of the mechanism for the preferential increase in the tumour RNA polymerase I

1980 ◽  
Vol 190 (3) ◽  
pp. 781-789 ◽  
Author(s):  
B W Duceman ◽  
S T Jacob
Keyword(s):  
Rna Polymerase ◽  
Tumour Growth ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Product Analysis ◽  
Sephadex Column ◽  
Tissue Homogenates ◽  
Enzyme Pattern ◽  
Polymerase I

The amount and/or activity of DNA-dependent RNA polymerase I, Ii and III from resting liver, regenerating liver and a series of Morris hepatomas (5123D, 7800, 7777, 3924A) were determined after extraction of the enzymes from whole tissue homogenates and subsequent fractionation by DEAE-Sephadex column chromatography. When compared with resting liver, the tumours exhibited a characteristic enzyme pattern in which polymerase I, but not II, was increased. The increase in RNA polymerase I was proportional to the tumour growth rates. Alterations in polymerase III were confined to the most rapidly proliferating hepatomas. By contrast, all classes of RNA polymerase were found to be increased during liver regeneration. Relative to resting liver, the fastest growing tumour, 3924A, exhibited the highest activities and/or amounts of RNA polymerase I (8-fold) and III (5-fold) per g of tissue. These alterations in the tumour RNA polymerases were reflected in corresponding increases in the transcriptionally active (bound or chromatin-associated) enzyme population. The mechanisms underlying the augmented synthesis of RNA in vitro by bound polymerase I from hepatoma 3924A were elucidated by product analysis. The results indicated that, relative to liver RNA polymerase I, the tumour enzyme produced more nascent RNA chains and elongated these chains at a faster rate. The number of 3'-termini, as measured by incorporation into uridine, was higher in the hepatoma even under conditions which prevented re-initiation. suggesting increased amount of transcriptionally active RNA polymerase I in the tumour.

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