scholarly journals Interaction of TATA-Binding Protein with Upstream Activation Factor Is Required for Activated Transcription of Ribosomal DNA by RNA Polymerase I in Saccharomyces cerevisiae In Vivo

1998 ◽  
Vol 18 (7) ◽  
pp. 3752-3761 ◽  
Author(s):  
Joan S. Steffan ◽  
Daniel A. Keys ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Ribosomal Dna ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Activation Factor ◽  
Polymerase I ◽  
Two Hybrid

ABSTRACT Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations inRRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast cells containing any one of these individual mutations,L110S, L269P, or L274Q, did not show any growth defects. However, cells containing a combination ofL110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo.

scholarly journals Casein Kinase 2 Associates with Initiation-Competent RNA Polymerase I and Has Multiple Roles in Ribosomal DNA Transcription

2006 ◽  
Vol 26 (16) ◽  
pp. 5957-5968 ◽  
Author(s):  
Tatiana B. Panova ◽  
Kostya I. Panov ◽  
Jackie Russell ◽  
Joost C. B. M. Zomerdijk
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Rna Polymerase I ◽  
Positive Effects ◽  
Pol I ◽  
Polymerase I

ABSTRACT Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Iβ isoform and not with Pol Iα. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Iβ-associated CK2 can phosphorylate topoisomerase IIα in Pol Iβ, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAFI110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAFI110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.

scholarly journals Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I

2019 ◽  
Vol 295 (5) ◽  
pp. 1288-1299 ◽  
Author(s):  
Catherine E. Scull ◽  
Andrew M. Clarke ◽  
Aaron L. Lucius ◽  
David Alan Schneider
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Dna Sequence ◽  
Transcription Elongation ◽  
Gc Content ◽  
Rna Polymerase I ◽  
Pol I ◽  
Polymerase I

The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo. Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.

scholarly journals TATA Binding Protein Can Stimulate Core-Directed Transcription by Yeast RNA Polymerase I

2000 ◽  
Vol 20 (14) ◽  
pp. 5269-5275 ◽  
Author(s):  
Pavel Aprikian ◽  
Beth Moorefield ◽  
Ronald H. Reeder
Keyword(s):  
Rna Polymerase ◽  
Binding Protein ◽  
Core Promoter ◽  
Contact Point ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Dual Function ◽  
Polymerase I

ABSTRACT The TATA binding protein (TBP) interacts with two transcription factor complexes, upstream activating factor (UAF) and core factor (CF), to direct transcription by RNA polymerase I (polI) in the yeastSaccharomyces cerevisiae. Previous work indicates that one function of TBP is to serve as a bridge, ennabling UAF to recruit and stabilize the binding of CF (23, 24). In this work we show that, in addition to aiding recruitment, TBP also directly aids CF function. Overexpression of TBP in strains with UAF components deleted will stimulate CF-directed transcription nearly to wild-type levels in vivo. In vitro, increasing the concentration of TBP stimulates CF-directed transcription in the absence of either UAF or its DNA binding site. This dual function of TBP, serving as a critical member of a core promoter complex as well as a contact point for upstream activators, appears similar to the dual roles that TBP also plays in transcription by RNA polII.

scholarly journals Saccharomyces cerevisiae RNA Polymerase I Terminates Transcription at the Reb1 Terminator In Vivo

1999 ◽  
Vol 19 (11) ◽  
pp. 7369-7376 ◽  
Author(s):  
Ronald H. Reeder ◽  
Palmira Guevara ◽  
Judith G. Roan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Rnase Iii ◽  
S1 Nuclease ◽  
Nuclease Mapping ◽  
Fail Safe ◽  
Polymerase I

ABSTRACT We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3′ end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3′ end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies.

Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals In vivo evaluation of the interaction between the Escherichia coli IGP synthase subunits using the Bacterial Two-Hybrid system

2020 ◽  
Vol 367 (14) ◽  
Author(s):  
Sofia Chioccioli ◽  
Patrizia Bogani ◽  
Sara Del Duca ◽  
Lara Mitia Castronovo ◽  
Alberto Vassallo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hybrid System ◽  
De Novo ◽  
Glycerol Phosphate ◽  
In Vivo Evaluation ◽  
Igp Synthase ◽  
Imidazole Glycerol Phosphate ◽  
Two Hybrid

ABSTRACT Histidine biosynthesis is one of the most characterized metabolic routes for its antiquity and its central role in cellular metabolism; indeed, it represents a cross-road between nitrogen metabolism and de novo synthesis of purines. This interconnection is due to the activity of imidazole glycerol phosphate synthase, a heterodimeric enzyme constituted by the products of two his genes, hisH and hisF, encoding a glutamine amidotransferase and a cyclase, respectively. Despite their interaction was suggested by several in vitro experiments, their in vivo complex formation has not been demonstrated. On the contrary, the analysis of the entire Escherichia coli interactome performed using the yeast two hybrid system did not suggest the in vivo interaction of the two IGP synthase subunits. The aim of this study was to demonstrate the interaction of the two proteins using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system. Data obtained demonstrated the in vivo interaction occurring between the proteins encoded by the E. coli hisH and hisF genes; this finding might also open the way to pharmaceutical applications through the design of selective drugs toward this enzyme.

scholarly journals Role of TATA Binding Protein (TBP) in Yeast Ribosomal DNA Transcription by RNA Polymerase I: Defects in the Dual Functions of Transcription Factor UAF Cannot Be Suppressed by TBP

2001 ◽  
Vol 21 (7) ◽  
pp. 2292-2297 ◽  
Author(s):  
Imran Siddiqi ◽  
John Keener ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Rrna Transcription ◽  
Pol I ◽  
Mutant Strains ◽  
Polymerase I

ABSTRACT Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I. We found previously that yeast strains carrying deletions in the UAF component RRN9switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription. These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth. Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P. Aprikian, B. Moorefield, and R. H. Reeder, Mol. Cell. Biol. 20:5269–5275, 2000). Here, we demonstrate that deletions in the UAF component RRN5,RRN9, or RRN10 lead to Pol II transcription of rDNA. TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription. We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP. In diploid strains lacking both copies of the UAF componentRRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I. These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.

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