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

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.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.754 ◽

1991 ◽

Vol 11 (2) ◽

pp. 754-764 ◽

Cited By ~ 42

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5640-5651.1992 ◽

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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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

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.754-764.1991 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5640 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5640-5651 ◽

Cited By ~ 105

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.114 ◽

1993 ◽

Vol 13 (1) ◽

pp. 114-122 ◽

Cited By ~ 59

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.114-122.1993 ◽

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.

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Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4702 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4702-4710 ◽

Cited By ~ 70

Author(s):

G V Shpakovski ◽

J Acker ◽

M Wintzerith ◽

J F Lacroix ◽

P Thuriaux ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Homo Sapiens ◽

Binding Domain ◽

Zinc Binding ◽

Carboxy Terminal ◽

Zinc Binding Domain ◽

Yeast Mutants ◽

Rna Polymerase Subunits

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit.

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