Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association

1992 ◽  
Vol 12 (3) ◽  
pp. 1087-1095
Author(s):  
M Werner ◽  
S Hermann-Le Denmat ◽  
I Treich ◽  
A Sentenac ◽  
P Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Zinc Binding ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Zinc Ions ◽  
Amino Terminal ◽  
Zinc Binding Domain ◽  
Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

scholarly journals Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.

1992 ◽  
Vol 12 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
M Werner ◽  
S Hermann-Le Denmat ◽  
I Treich ◽  
A Sentenac ◽  
P Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Zinc Binding ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Zinc Ions ◽  
Amino Terminal ◽  
Zinc Binding Domain ◽  
Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

scholarly journals The zinc-binding motif in tankyrases is required for the structural integrity and proper function of the catalytic domain

2021 ◽  
Author(s):  
Sven T Sowa ◽  
Lari Lehtiö
Keyword(s):  
Structural Integrity ◽  
Catalytic Domain ◽  
Zinc Binding ◽  
Structural Defects ◽  
Proper Function ◽  
Binding Motif ◽  
Acceptor Site ◽  
Structural Element ◽  
Zinc Binding Motif

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and they are therefore possible drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyl-transferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on activity, stability and structure of human tankyrases. We generated mutants of human TNKS1 and TNKS2 abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive likely due to introduction of major structural defects, the TNKS2 mutant remained active and displayed a different activity profile compared to the wild type.

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 The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination

2000 ◽  
Vol 14 (6) ◽  
pp. 731-739
Author(s):  
Randolph S. Watnick ◽  
Stephanie Chiyoko Herring ◽  
Arthur G. Palmer ◽  
Max E. Gottesman
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Tryptophan Residue ◽  
Binding Motif ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage λ nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun–RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data.

scholarly journals Phosphonamidates are the first phosphorus-based zinc binding motif to show inhibition of β-class carbonic anhydrases from bacteria, fungi, and protozoa

2019 ◽  
Vol 35 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Siham A. Alissa ◽  
Hanan A. Alghulikah ◽  
Zeid A. Alothman ◽  
Sameh M. Osman ◽  
Sonia Del Prete ◽  
...  
Keyword(s):  
Zinc Binding ◽  
Binding Motif ◽  
Carbonic Anhydrases ◽  
Zinc Binding Motif

Conformational dynamics and allosteric effect modulated by the unique zinc-binding motif in class IIa HDACs

2019 ◽  
Vol 21 (23) ◽  
pp. 12173-12183 ◽  
Author(s):  
Huawei Liu ◽  
Fan Zhang ◽  
Kai Wang ◽  
Xiaowen Tang ◽  
Ruibo Wu
Keyword(s):  
Histone Deacetylases ◽  
Conformational Dynamics ◽  
Zinc Binding ◽  
Binding Motif ◽  
Allosteric Effect ◽  
Class Iia Hdacs ◽  
Zinc Binding Motif

Class IIa histone deacetylases (HDACs) have been considered as potential targets for the treatment of several diseases.

scholarly journals Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

2001 ◽  
Vol 358 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Henning B. BOLDT ◽  
Michael T. OVERGAARD ◽  
Lisbeth S. LAURSEN ◽  
Kathrin WEYER ◽  
Lars SOTTRUP-JENSEN ◽  
...  
Keyword(s):  
Plasma Protein ◽  
Active Site ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Protein A ◽  
Residual Activity ◽  
Zinc Binding ◽  
Binding Motif ◽  
Dependent Manner ◽  
Zinc Binding Motif

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

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