scholarly journals Gene RRN4 in Saccharomyces cerevisiae encodes the A12.2 subunit of RNA polymerase I and is essential only at high temperatures.

1993 ◽  
Vol 13 (1) ◽  
pp. 114-122 ◽  
Author(s):  
Y Nogi ◽  
R Yano ◽  
J Dodd ◽  
C Carles ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Temperature Sensitive ◽  
Polymerase I ◽  
Temperature Sensitive Phenotype ◽  
Null Mutants

We have previously isolated mutants of Saccharomyces cerevisiae that are primarily defective in transcription of 35S rRNA genes by RNA polymerase I and have identified genes (RRN1 to RRN9) involved in this process. We have now cloned the RRN4 gene by complementation of the temperature-sensitive phenotype of the rrn4-1 mutant and have determined its complete nucleotide sequence. The following results demonstrate that the RRN4 gene encodes the A12.2 subunit of RNA polymerase I. First, RRN4 protein expressed in Escherichia coli reacted with a specific antiserum against A12.2. Second, amino acid sequences of three tryptic peptides obtained from A12.2 were determined, and these sequences are found in the deduced amino acid sequence of the RRN4 protein. The amino acid sequence of the RRN4 protein (A12.2) is similar to that of the RPB9 (B12.6) subunit of yeast RNA polymerase II; the similarity includes the presence of two putative zinc-binding domains. Thus, A12.2 is a homolog of B12.6. We propose to rename the RRN4 gene RPA12. Deletion of RPA12 produces cells that are heat but not cold sensitive for growth. We have found that in such null mutants growing at permissive temperatures, the cellular concentration of A190, the largest subunit of RNA polymerase I, is lower than in the wild type. In addition, the temperature-sensitive phenotype of the rpa12 null mutants can be partially suppressed by RPA190 (the gene for A190) on multicopy plasmids. These results suggest that A12.2 plays a role in the assembly of A190 into a stable polymerase I structure.

Gene RRN4 in Saccharomyces cerevisiae encodes the A12.2 subunit of RNA polymerase I and is essential only at high temperatures

1993 ◽  
Vol 13 (1) ◽  
pp. 114-122
Author(s):  
Y Nogi ◽  
R Yano ◽  
J Dodd ◽  
C Carles ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Temperature Sensitive ◽  
Polymerase I ◽  
Temperature Sensitive Phenotype ◽  
Null Mutants

We have previously isolated mutants of Saccharomyces cerevisiae that are primarily defective in transcription of 35S rRNA genes by RNA polymerase I and have identified genes (RRN1 to RRN9) involved in this process. We have now cloned the RRN4 gene by complementation of the temperature-sensitive phenotype of the rrn4-1 mutant and have determined its complete nucleotide sequence. The following results demonstrate that the RRN4 gene encodes the A12.2 subunit of RNA polymerase I. First, RRN4 protein expressed in Escherichia coli reacted with a specific antiserum against A12.2. Second, amino acid sequences of three tryptic peptides obtained from A12.2 were determined, and these sequences are found in the deduced amino acid sequence of the RRN4 protein. The amino acid sequence of the RRN4 protein (A12.2) is similar to that of the RPB9 (B12.6) subunit of yeast RNA polymerase II; the similarity includes the presence of two putative zinc-binding domains. Thus, A12.2 is a homolog of B12.6. We propose to rename the RRN4 gene RPA12. Deletion of RPA12 produces cells that are heat but not cold sensitive for growth. We have found that in such null mutants growing at permissive temperatures, the cellular concentration of A190, the largest subunit of RNA polymerase I, is lower than in the wild type. In addition, the temperature-sensitive phenotype of the rpa12 null mutants can be partially suppressed by RPA190 (the gene for A190) on multicopy plasmids. These results suggest that A12.2 plays a role in the assembly of A190 into a stable polymerase I structure.

scholarly journals Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

1991 ◽  
Vol 11 (2) ◽  
pp. 754-764 ◽  
Author(s):  
R Yano ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Temperature Sensitive ◽  
Polymerase I ◽  
Zinc Binding Domain

The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.

Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I

1991 ◽  
Vol 11 (2) ◽  
pp. 754-764
Author(s):  
R Yano ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Temperature Sensitive ◽  
Polymerase I ◽  
Zinc Binding Domain

The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.

scholarly journals I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

1998 ◽  
Vol 18 (10) ◽  
pp. 5809-5817 ◽  
Author(s):  
Jue Lin ◽  
Volker M. Vogt
Keyword(s):  
Rna Polymerase ◽  
Homing Endonuclease ◽  
Group I Intron ◽  
Subcellular Fractionation ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Temperature Sensitive ◽  
Group I ◽  
Polymerase I ◽  
Mutant Forms

ABSTRACT PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023–1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as theTetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.

scholarly journals A new marker sequence for systematics of medically important fungi based on amino acid sequence of the largest subunit of RNA polymerase I

2016 ◽  
pp. myw098
Author(s):  
Chiaki Yamanishi ◽  
Mohamed Mahdi Alshahni ◽  
Ayako Sano ◽  
Ikuo Nakamura ◽  
Koichi Makimura
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Polymerase I

Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (12) ◽  
pp. 5640-5651
Author(s):  
R Yano ◽  
M Oakes ◽  
M Yamaghishi ◽  
J A Dodd ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Temperature Sensitive ◽  
Pol I ◽  
Polymerase I ◽  
Zinc Binding Domain

The SRP1-1 mutation is an allele-specific dominant suppressor of temperature-sensitive mutations in the zinc-binding domain of the A190 subunit of Saccharomyces cerevisiae RNA polymerase I (Pol I). We found that it also suppresses temperature-sensitive mutations in the zinc-binding domain of the Pol I A135 subunit. This domain had been suggested to be in physical proximity to the A190 zinc-binding domain. We have cloned the SRP1 gene and determined its nucleotide sequence. The gene encodes a protein of 542 amino acids consisting of three domains: the central domain, which is composed of eight (degenerate) 42-amino-acid contiguous tandem repeats, and the surrounding N-terminal and C-terminal domains, both of which contain clusters of acidic and basic amino acids and are very hydrophilic. The mutational alteration (P219Q) responsible for the suppression was found to be in the central domain. Using antibody against the SRP1 protein, we have found that SRP1 is mainly localized at the periphery of the nucleus, apparently more concentrated in certain regions, as suggested by a punctate pattern in immunofluorescence microscopy. We suggest that SRP1 is a component of a larger macromolecular complex associated with the nuclear envelope and interacts with Pol I either directly or indirectly through other components in the structure containing SRP1.

scholarly journals Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes.

1991 ◽  
Vol 11 (2) ◽  
pp. 746-753 ◽  
Author(s):  
J H McCusker ◽  
M Yamagishi ◽  
J M Kolb ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Gene Dosage ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Polymerase I ◽  
Zinc Binding Domain

Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations. All the SRP mutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants. SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations. All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5. In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion. Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.

Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes

1991 ◽  
Vol 11 (2) ◽  
pp. 746-753 ◽  
Author(s):  
J H McCusker ◽  
M Yamagishi ◽  
J M Kolb ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Gene Dosage ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Polymerase I ◽  
Zinc Binding Domain

Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations. All the SRP mutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants. SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations. All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5. In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion. Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.

scholarly journals RNA Polymerase I Transcription Factors in Active Yeast rRNA Gene Promoters Enhance UV Damage Formation and Inhibit Repair

2005 ◽  
Vol 25 (5) ◽  
pp. 1586-1595 ◽  
Author(s):  
Andreas Meier ◽  
Fritz Thoma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Stable Complex ◽  
Rrna Gene ◽  
Gene Promoters ◽  
The Core ◽  
Polymerase I

ABSTRACT UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.

scholarly journals Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (12) ◽  
pp. 5640-5651 ◽  
Author(s):  
R Yano ◽  
M Oakes ◽  
M Yamaghishi ◽  
J A Dodd ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Temperature Sensitive ◽  
Pol I ◽  
Polymerase I ◽  
Zinc Binding Domain

The SRP1-1 mutation is an allele-specific dominant suppressor of temperature-sensitive mutations in the zinc-binding domain of the A190 subunit of Saccharomyces cerevisiae RNA polymerase I (Pol I). We found that it also suppresses temperature-sensitive mutations in the zinc-binding domain of the Pol I A135 subunit. This domain had been suggested to be in physical proximity to the A190 zinc-binding domain. We have cloned the SRP1 gene and determined its nucleotide sequence. The gene encodes a protein of 542 amino acids consisting of three domains: the central domain, which is composed of eight (degenerate) 42-amino-acid contiguous tandem repeats, and the surrounding N-terminal and C-terminal domains, both of which contain clusters of acidic and basic amino acids and are very hydrophilic. The mutational alteration (P219Q) responsible for the suppression was found to be in the central domain. Using antibody against the SRP1 protein, we have found that SRP1 is mainly localized at the periphery of the nucleus, apparently more concentrated in certain regions, as suggested by a punctate pattern in immunofluorescence microscopy. We suggest that SRP1 is a component of a larger macromolecular complex associated with the nuclear envelope and interacts with Pol I either directly or indirectly through other components in the structure containing SRP1.

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