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

1995 ◽  
Vol 15 (9) ◽  
pp. 4702-4710 ◽  
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.

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.

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.

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 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 Farnesylation of Ydj1 Is Required for In Vivo Interaction with Hsp90 Client Proteins

2008 ◽  
Vol 19 (12) ◽  
pp. 5249-5258 ◽  
Author(s):  
Gary A. Flom ◽  
Marta Lemieszek ◽  
Elizabeth A. Fortunato ◽  
Jill L. Johnson
Keyword(s):  
Molecular Chaperone ◽  
Steroid Receptors ◽  
Binding Domain ◽  
Zinc Binding ◽  
Physical Interaction ◽  
Client Proteins ◽  
Carboxy Terminal ◽  
Zinc Binding Domain ◽  
Substrate Binding Domain

Ydj1 of Saccharomyces cerevisiae is an abundant cytosolic Hsp40, or J-type, molecular chaperone. Ydj1 cooperates with Hsp70 of the Ssa family in the translocation of preproteins to the ER and mitochondria and in the maturation of Hsp90 client proteins. The substrate-binding domain of Ydj1 directly interacts with steroid receptors and is required for the activity of diverse Hsp90-dependent client proteins. However, the effect of Ydj1 alteration on client interaction was unknown. We analyzed the in vivo interaction of Ydj1 with the protein kinase Ste11 and the glucocorticoid receptor. Amino acid alterations in the proposed client-binding domain or zinc-binding domain had minor effects on the physical interaction of Ydj1 with both clients. However, alteration of the carboxy-terminal farnesylation signal disrupted the functional and physical interaction of Ydj1 and Hsp90 with both clients. Similar effects were observed upon deletion of RAM1, which encodes one of the subunits of yeast farnesyltransferase. Our results indicate that farnesylation is a major factor contributing to the specific requirement for Ydj1 in promoting proper regulation and activation of diverse Hsp90 clients.

scholarly journals Structural basis of bacterial σ 28 ‐mediated transcription reveals roles of the RNA polymerase zinc‐binding domain

2020 ◽  
Vol 39 (14) ◽  
Author(s):  
Wei Shi ◽  
Wei Zhou ◽  
Baoyue Zhang ◽  
Shaojia Huang ◽  
Yanan Jiang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Domain ◽  
Zinc Binding ◽  
Structural Basis ◽  
Zinc Binding Domain
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