scholarly journals Additional RNA polymerase I initiation site within the nontranscribed spacer region of the rat rRNA gene.

1987 ◽  
Vol 7 (7) ◽  
pp. 2388-2396 ◽  
Author(s):  
B G Cassidy ◽  
H F Yang-Yen ◽  
L I Rothblum
Keyword(s):  
Rna Polymerase ◽  
Southern Hybridization ◽  
Initiation Site ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Nontranscribed Spacer ◽  
Upstream Promoter ◽  
Polymerase I

We identified and characterized an additional promoter within the nontranscribed spacer (NTS) of the rat ribosomal gene repeat that is capable of supporting initiation of transcription by RNA polymerase I in vitro. Within this promoter there is a sequence of 13 nucleotides which is 100% homologous to nucleotides -18 to -6 (+1 being the first nucleotide of 45S rRNA) of the major promoter of 45S pre-rRNA and is located between nucleotides -731 and -719. To identify the exact location of the upstream initiation site, the RNA synthesized in vitro from this new promoter was gel isolated and subjected to fingerprint analysis, Southern hybridization, and reverse transcriptase elongation. Based on these analyses, the in vitro-synthesized RNA initiates with an A at nucleotide -713. When compared individually, the upstream promoter was transcribed ninefold less efficiently than the major promoter. When templates which contain both promoters on the same piece of DNA were transcribed, the major promoter was at least 50-fold more efficient.

Additional RNA polymerase I initiation site within the nontranscribed spacer region of the rat rRNA gene

1987 ◽  
Vol 7 (7) ◽  
pp. 2388-2396
Author(s):  
B G Cassidy ◽  
H F Yang-Yen ◽  
L I Rothblum
Keyword(s):  
Rna Polymerase ◽  
Southern Hybridization ◽  
Initiation Site ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Nontranscribed Spacer ◽  
Upstream Promoter ◽  
Polymerase I

We identified and characterized an additional promoter within the nontranscribed spacer (NTS) of the rat ribosomal gene repeat that is capable of supporting initiation of transcription by RNA polymerase I in vitro. Within this promoter there is a sequence of 13 nucleotides which is 100% homologous to nucleotides -18 to -6 (+1 being the first nucleotide of 45S rRNA) of the major promoter of 45S pre-rRNA and is located between nucleotides -731 and -719. To identify the exact location of the upstream initiation site, the RNA synthesized in vitro from this new promoter was gel isolated and subjected to fingerprint analysis, Southern hybridization, and reverse transcriptase elongation. Based on these analyses, the in vitro-synthesized RNA initiates with an A at nucleotide -713. When compared individually, the upstream promoter was transcribed ninefold less efficiently than the major promoter. When templates which contain both promoters on the same piece of DNA were transcribed, the major promoter was at least 50-fold more efficient.

scholarly journals RNA polymerase I–specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle

2011 ◽  
Vol 192 (2) ◽  
pp. 277-293 ◽  
Author(s):  
Benjamin Albert ◽  
Isabelle Léger-Silvestre ◽  
Christophe Normand ◽  
Martin K. Ostermaier ◽  
Jorge Pérez-Fernández ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Loading Rate ◽  
Gene Copy Number ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Gene Copy ◽  
Rrna Gene ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I

RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

scholarly journals In vitro definition of the yeast RNA polymerase I enhancer

1993 ◽  
Vol 13 (5) ◽  
pp. 2644-2654
Author(s):  
M C Schultz ◽  
S Y Choe ◽  
R H Reeder
Keyword(s):  
Rna Polymerase ◽  
Core Promoter ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Stable Complex ◽  
Promoter Complex ◽  
Definition Of ◽  
Polymerase I

In vitro conditions are reported under which an EcoRI-HpaI fragment of the Saccharomyces cerevisiae ribosomal gene spacer will enhance transcription from an adjacent RNA polymerase I promoter. Enhancement is largely independent of orientation and distance and is proportional to copy number. Mapping experiments reveal that two separate regions of the EcoRI-HpaI fragment are independently capable of promoter stimulation. These regions appear to correspond to elements which have been shown by previous workers to cause enhancement in vivo. Using the detergent Sarkosyl to limit the number of rounds of transcription from each promoter, we found that the degree of enhancement is similar whether one or many rounds of transcription occur. This finding supports a model in which the enhancer increases the number of stable promoter complexes but does not alter the loading of polymerase on an active promoter. Once the stable promoter complex is formed, the enhancer can be physically severed from the promoter with no loss of enhancement. Likewise, the upstream activation region of the promoter can be severed from the core promoter domain once the stable complex has been formed. These results are interpreted to mean that the enhancer functions only to assist stable complex formation and, once that is accomplished, the enhancer is dispensable.

scholarly journals Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

1994 ◽  
Vol 14 (7) ◽  
pp. 5010-5021 ◽  
Author(s):  
R P Brun ◽  
K Ryan ◽  
B Sollner-Webb
Keyword(s):  
Rna Polymerase ◽  
Critical Distance ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Transcription Process ◽  
Factor C ◽  
Polymerase I

Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates (NTPs). By using 3'dNTPs to specifically halt elongation, C* is seen to remain active through transcription complex assembly, initiation, and the first approximately 37 nucleotides of elongation, but it is inactivated before synthesis proceeds beyond approximately 40 nucleotides. When elongation is halted before this critical distance, the C* remains active and on that template complex, greatly extending the kinetics of transcription and generating manyfold more transcripts than would have been synthesized if elongation had proceeded past the critical distance where C* is inactivated. In complementary in vivo analysis under conditions where C* activity is not replenished, C* activity becomes depleted from cells, but this also occurs only when there is ongoing rDNA transcription. Thus, both in vitro and in vivo, the specific initiation-conferring component of the RNA polymerase I holoenzyme is used stoichiometrically in the transcription process.

scholarly journals The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3.

1997 ◽  
Vol 17 (10) ◽  
pp. 6175-6183 ◽  
Author(s):  
C Cadwell ◽  
H J Yoon ◽  
Y Zebarjadian ◽  
J Carbon
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Genomic Library ◽  
Rna Polymerase I ◽  
Nucleolar Protein ◽  
Rrna Gene ◽  
Temperature Sensitive ◽  
Cell Biol ◽  
Polymerase I

Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W. Jiang, K. Middleton, H.-J. Yoon, C. Fouquet, and J. Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolar-cytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperature-sensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5delta null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.

scholarly journals Formation of the transcription initiation complex on mammalian rDNA.

1986 ◽  
Vol 6 (10) ◽  
pp. 3418-3427 ◽  
Author(s):  
H Kato ◽  
M Nagamine ◽  
R Kominami ◽  
M Muramatsu
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Short Pulse ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Preinitiation Complex ◽  
Initiation Complex ◽  
Transcription Initiation Complex ◽  
Polymerase I

Steps for the formation of transcription initiation complex on the human rRNA gene (rDNA) in vitro were analyzed with partially purified transcription factors and RNA polymerase I. The reaction requires at least two factors besides RNA polymerase I for maximal efficiency. Preincubation and short-pulse analyses of the accurate transcripts revealed the following steps. First, the species-dependent factor, designated TFID, bound to the rDNA template, forming a preinitiation complex (PIC-1) which was resistant to a moderate concentration (0.015 to 0.02%) of Sarkosyl. Other factors, designated TFIA and RNA polymerase I, were then added to convert it to the final preinitiation complex PIC-3. This complex incorporated the first two nucleoside triphosphates of the starting site to complete the initiation complex (IC), which was resistant to a high concentration (0.2%) of Sarkosyl. Binding of TFID was rate limiting in the overall initiation reaction in vitro. Together with the kinetics of incorporation, the results are interpreted to mean that TFID, one bound, remains complexed with rDNA together with TFIA as the PIC-2 for many rounds of transcription by RNA polymerase I. Thus, the formation of PIC-2 may be a prerequisite for the stable opening of rDNA for transcription in vivo.

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process

1994 ◽  
Vol 14 (7) ◽  
pp. 5010-5021
Author(s):  
R P Brun ◽  
K Ryan ◽  
B Sollner-Webb
Keyword(s):  
Rna Polymerase ◽  
Critical Distance ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Transcription Process ◽  
Factor C ◽  
Polymerase I

Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates (NTPs). By using 3'dNTPs to specifically halt elongation, C* is seen to remain active through transcription complex assembly, initiation, and the first approximately 37 nucleotides of elongation, but it is inactivated before synthesis proceeds beyond approximately 40 nucleotides. When elongation is halted before this critical distance, the C* remains active and on that template complex, greatly extending the kinetics of transcription and generating manyfold more transcripts than would have been synthesized if elongation had proceeded past the critical distance where C* is inactivated. In complementary in vivo analysis under conditions where C* activity is not replenished, C* activity becomes depleted from cells, but this also occurs only when there is ongoing rDNA transcription. Thus, both in vitro and in vivo, the specific initiation-conferring component of the RNA polymerase I holoenzyme is used stoichiometrically in the transcription process.

Formation of the transcription initiation complex on mammalian rDNA

1986 ◽  
Vol 6 (10) ◽  
pp. 3418-3427
Author(s):  
H Kato ◽  
M Nagamine ◽  
R Kominami ◽  
M Muramatsu
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Short Pulse ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Preinitiation Complex ◽  
Initiation Complex ◽  
Transcription Initiation Complex ◽  
Polymerase I

Steps for the formation of transcription initiation complex on the human rRNA gene (rDNA) in vitro were analyzed with partially purified transcription factors and RNA polymerase I. The reaction requires at least two factors besides RNA polymerase I for maximal efficiency. Preincubation and short-pulse analyses of the accurate transcripts revealed the following steps. First, the species-dependent factor, designated TFID, bound to the rDNA template, forming a preinitiation complex (PIC-1) which was resistant to a moderate concentration (0.015 to 0.02%) of Sarkosyl. Other factors, designated TFIA and RNA polymerase I, were then added to convert it to the final preinitiation complex PIC-3. This complex incorporated the first two nucleoside triphosphates of the starting site to complete the initiation complex (IC), which was resistant to a high concentration (0.2%) of Sarkosyl. Binding of TFID was rate limiting in the overall initiation reaction in vitro. Together with the kinetics of incorporation, the results are interpreted to mean that TFID, one bound, remains complexed with rDNA together with TFIA as the PIC-2 for many rounds of transcription by RNA polymerase I. Thus, the formation of PIC-2 may be a prerequisite for the stable opening of rDNA for transcription in vivo.

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