DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.

Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.

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Cell Cycle Localization, Dimerization, and Binding Domain Architecture of the Telomere Protein cPot1

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.2091-2102.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 2091-2102 ◽

Cited By ~ 28

Author(s):

Chao Wei ◽

Carolyn M. Price

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Binding Site ◽

Domain Architecture ◽

Binding Domain ◽

Binding Motif ◽

Dna Binding Domain ◽

Telomere Protein ◽

Ob Fold

ABSTRACT Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.

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A position-dependent transcription-activating domain in TFIIIA

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7496-7506.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7496-7506

Author(s):

X Mao ◽

M K Darby

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Zinc Finger ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Rna Polymerase Iii ◽

Binding Domain ◽

Dna Binding Domain ◽

5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

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A position-dependent transcription-activating domain in TFIIIA.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7496 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7496-7506 ◽

Cited By ~ 15

Author(s):

X Mao ◽

M K Darby

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Zinc Finger ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Rna Polymerase Iii ◽

Binding Domain ◽

Dna Binding Domain ◽

5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

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Purification and Characterization of the AAA+ Domain of Sinorhizobium meliloti DctD, a σ54-Dependent Transcriptional Activator

Journal of Bacteriology ◽

10.1128/jb.186.11.3499-3507.2004 ◽

2004 ◽

Vol 186 (11) ◽

pp. 3499-3507 ◽

Cited By ~ 22

Author(s):

Hao Xu ◽

Baohua Gu ◽

B. Tracy Nixon ◽

Timothy R. Hoover

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Sinorhizobium Meliloti ◽

Atp Hydrolysis ◽

Binding Domain ◽

Stable Complex ◽

Dna Binding Domain ◽

Dna Binding Domains ◽

Binding Domains

ABSTRACT Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD( Δ 1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD( Δ 1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD( Δ 1-142) displayed multiple species. The DctD AAA+ domain, but not DctD( Δ 1-142), formed a stable complex with σ54 in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with σ54 in addition to its role in recognition of upstream activation sequences.

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Interaction with free β′ subunit unmasks DNA-binding domain of RNA polymerase σ subunit

FEBS Letters ◽

10.1016/s0014-5793(99)00778-4 ◽

1999 ◽

Vol 454 (1-2) ◽

pp. 71-74 ◽

Cited By ~ 13

Author(s):

Andrey Kulbachinskiy ◽

Arkady Mustaev ◽

Alex Goldfarb ◽

Vadim Nikiforov

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Binding Domain ◽

Dna Binding Domain ◽

Β Subunit

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A bipartite DNA-binding domain in yeast Reb1p.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1173 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1173-1182 ◽

Cited By ~ 33

Author(s):

B E Morrow ◽

Q Ju ◽

J R Warner

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Kluyveromyces Lactis ◽

Binding Domain ◽

Dna Binding Domain ◽

Coding Region ◽

Active Growth ◽

Yeast Saccharomyces Cerevisiae

The REB1 gene encodes a DNA-binding protein (Reb1p) that is essential for growth of the yeast Saccharomyces cerevisiae. Reb1p binds to sites within transcriptional control regions of genes transcribed by either RNA polymerase I or RNA polymerase II. The sequence of REB1 predicts a protein of 809 amino acids. To define the DNA-binding domain of Reb1p, a series of 5' and 3' deletions within the coding region was constructed in a bacterial expression vector. Analysis of the truncated Reb1p proteins revealed that nearly 400 amino acids of the C-terminal portion of the protein are required for maximal DNA-binding activity. To further define the important structural features of Reb1p, the REB1 homolog from a related yeast, Kluyveromyces lactis, was cloned by genetic complementation. The K. lactis REB1 gene supports active growth of an S. cerevisiae strain whose REB1 gene has been deleted. The Reb1p proteins of the two organisms generate almost identical footprints on DNA, yet the K. lactis REB1 gene encodes a polypeptide of only 595 amino acids. Comparison of the two Reb1p sequences revealed that within the region necessary for the binding of Reb1p to DNA were two long regions of nearly perfect identity, separated in the S. cerevisiae Reb1p by nearly 150 amino acids but in the K. lactis Reb1p by only 40 amino acids. The first includes a 105-amino-acid region related to the DNA-binding domain of the myb oncoprotein; the second bears a faint resemblance to myb. The hypothesis that the DNA-binding domain of Reb1p is formed from these two conserved regions was confirmed by deletion of as many as 90 amino acids between them, with little effect on the DNA-binding ability of the resultant protein. We suggest that the DNA-binding domain of Reb1p is made up of two myb-like regions that, unlike myb itself, are separated by as many as 150 amino acids. Since Reb1p protects only 15 to 20 nucleotides in a chemical or enzymatic footprint assay, the protein must fold such that the two components of the binding site are adjacent.

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Helicobacter pylori FlgR Is an Enhancer-Independent Activator of σ54-RNA Polymerase Holoenzyme

Journal of Bacteriology ◽

10.1128/jb.186.14.4535-4542.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4535-4542 ◽

Cited By ~ 46

Author(s):

Priyanka Brahmachary ◽

Mona G. Dashti ◽

Jonathan W. Olson ◽

Timothy R. Hoover

Keyword(s):

Helicobacter Pylori ◽

Dna Binding ◽

Rna Polymerase ◽

Bacterial Species ◽

Binding Domain ◽

Dna Binding Domain ◽

Amino Terminal ◽

H Pylori ◽

Carboxy Terminal ◽

Rna Polymerase Holoenzyme

ABSTRACT Helicobacter pylori FlgR activates transcription with σ54-RNA polymerase holoenzyme (σ54-holoenzyme) from at least five flagellar operons. Activators of σ54-holoenzyme generally bind enhancer sequences located >70 bp upstream of the promoter and contact σ54-holoenzyme bound at the promoter through DNA looping to activate transcription. H. pylori FlgR lacks the carboxy-terminal DNA-binding domain present in most σ54-dependent activators. As little as 42 bp of DNA upstream of the flaB promoter and 26 bp of DNA sequence downstream of the transcriptional start site were sufficient for efficient FlgR-mediated expression from a flaB′-′xylE reporter gene in H. pylori, indicating that FlgR does not use an enhancer to activate transcription. Other examples of σ54-dependent activators that lack a DNA-binding domain include Chlamydia trachomatis CtcC and activators from the other Chlamydia spp. whose genomes have been sequenced. FlgR from Helicobacter hepaticus and Campylobacter jejuni, which are closely related to H. pylori, appear to have carboxy-terminal DNA-binding domains, suggesting that the loss of the DNA-binding domain from H. pylori FlgR occurred after the divergence of these bacterial species. Removal of the amino-terminal regulatory domain of FlgR resulted in a constitutively active form of the protein that activated transcription from σ54-dependent genes in Escherichia coli. The truncated FlgR protein also activated transcription with E. coli σ54-holoenzyme in an in vitro transcription assay.

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