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 Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

2000 ◽  
Vol 20 (1) ◽  
pp. 104-112 ◽  
Author(s):  
Christine R. Rodriguez ◽  
Eun-Jung Cho ◽  
Michael-C. Keogh ◽  
Claire L. Moore ◽  
Arno L. Greenleaf ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Capping Enzyme ◽  
Carboxy Terminal ◽  
Polyadenylation Factor ◽  
Enzyme Targeting

ABSTRACT The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase–CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, whilesrb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

scholarly journals BRCA1 Can Modulate RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Levels

2004 ◽  
Vol 24 (16) ◽  
pp. 6947-6956 ◽  
Author(s):  
Annie Moisan ◽  
Chantal Larochelle ◽  
Benoît Guillemette ◽  
Luc Gaudreau
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Region ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Hcc1937 Cell ◽  
Carboxy Terminal ◽  
Cycle Regulation ◽  
Ctd Phosphorylation

ABSTRACT A high incidence of breast and ovarian cancers has been linked to mutations in the BRCA1 gene. BRCA1 has been shown to be involved in both positive and negative regulation of gene activity as well as in numerous other processes such as DNA repair and cell cycle regulation. Since modulation of the RNA polymerase II carboxy-terminal domain (CTD) phosphorylation levels could constitute an interface to all these functions, we wanted to directly test the possibility that BRCA1 might regulate the phosphorylation state of the CTD. We have shown that the BRCA1 C-terminal region can negatively modulate phosphorylation levels of the RNA polymerase II CTD by the Cdk-activating kinase (CAK) in vitro. Interestingly, the BRCA1 C-terminal region can directly interact with CAK and inhibit CAK activity by competing with ATP. Finally, we demonstrated that full-length BRCA1 can inhibit CTD phosphorylation when introduced in the BRCA1−/− HCC1937 cell line. Our results suggest that BRCA1 could play its ascribed roles, at least in part, by modulating CTD kinase components.

scholarly journals What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry

2021 ◽  
Author(s):  
Blase Matthew LeBlanc ◽  
Rosamaria Yvette Moreno ◽  
Edwin Escobar ◽  
Mukesh Kumar Venkat Ramani ◽  
Jennifer S Brodbelt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphorylation State ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Protein Encoding ◽  
Small Nuclear Rnas ◽  
Rnap Ii ◽  
Carboxy Terminal ◽  
Encoding Genes

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...

Study on the association between CTD peptides and zinc(ii)-dipicolylamine appended beta-cyclodextrin

RSC Advances ◽  
2015 ◽  
Vol 5 (98) ◽  
pp. 80434-80440 ◽  
Author(s):  
Saihui Zhang ◽  
Yantao Shi ◽  
Wei Wang ◽  
Zhi Yuan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Beta Cyclodextrin ◽  
Carboxy Terminal

Association between zinc(ii)-dipicolylamine appended beta-cyclodextrin and CTD (carboxy-terminal domain of RNA polymerase II) peptides with different phosphorylation patterns was studied by ITC and NMR.

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.

Sign in / Sign up

Export Citation Format

Share Document