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M R Paule

Publications and source records attributed to M R Paule.

At least 19 recordsLinked to original sources
PubMed2005

Mapping and functional characterization of the TAF11 interaction with TFIIA.

TFIIA interacts with TFIID via association with TATA binding protein (TBP) and TBP-associated factor 11 (TAF11). We previously identified a mutation in the small subunit of TFIIA (toa2-I27K) that is defective for interaction with TAF11. To further explore the functional link between TFIIA and TAF11, the toa2-I27K allele was utilized in a genetic screen to isolate compensatory mutants in TAF11. Analysis of these compensatory mutants revealed that the interaction between TAF11 and TFIIA involves two distinct regions of TAF11: the highly conserved histone fold domain and the N-terminal region. Cells expressing a TAF11 allele defective for interaction with TFIIA exhibit conditional growth phenotypes and defects in transcription. Moreover, TAF11 imparts changes to both TFIIA-DNA and TBP-DNA contacts in the context of promoter DNA. These alterations appear to enhance the formation and stabilization of the TFIIA-TBP-DNA complex. Taken together, these studies provide essential information regarding the molecular organization of the TAF11-TFIIA interaction and define a mechanistic role for this association in the regulation of gene expression in vivo.

Gene Expression Regulation, Fungal↗
PubMed2002

DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.

Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.

Acanthamoeba↗
PubMed2000

DNA melting and promoter clearance by eukaryotic RNA polymerase I.

Ribosomal RNA transcription initiation requires the melting of DNA to form an open complex, formation of the first few phosphodiester bonds, commencement of RNA polymerase I movement along the DNA, clearance of the promoter, and the formation of a steady-state ternary elongation complex. We examined DNA melting and promoter clearance by using potassium permanganate, diethylpyrocarbonate and methidiumpropylEDTA.Fe(II) footprinting. In combination, these methods demonstrated: (1) TIF-IB and RNA polymerase I are the only proteins required for formation of an initial approximately 9 base-pair open promoter region. This finding contradicts earlier results using diethylpyrocarbonate alone, which suggested an RNA synthesis requirement for stable melting. (2) DNA melting is temperature-dependent, with a tm between 15 and 20 degrees C. (3) Temperature-dependency of melting, as well as stalling the polymerase at sites close to the transcription start site revealed that the melted DNA region initially opens upstream of the transcription initiation site, and enlarges in a downstream direction coordinate with initiation, eventually attaining a steady-state transcription bubble of approximately 19 base-pairs. (4) The RNA-DNA hybrid protects the template DNA from single-strand footprinting reagents. The hybrid is 9 bp in length, consistent with the longer hybrid estimated by some for the Escherichia coli polymerase and with the hybrids estimated for eukaryotic polymerases II and III.

Acanthamoeba↗
PubMed2000

Survey and summary: transcription by RNA polymerases I and III.

The task of transcribing nuclear genes is shared between three RNA polymerases in eukaryotes: RNA polymerase (pol) I synthesizes the large rRNA, pol II synthesizes mRNA and pol III synthesizes tRNA and 5S rRNA. Although pol II has received most attention, pol I and pol III are together responsible for the bulk of transcriptional activity. This survey will summarise what is known about the process of transcription by pol I and pol III, how it happens and the proteins involved. Attention will be drawn to the similarities between the three nuclear RNA polymerase systems and also to their differences.

Animals↗
PubMed1998

A novel transcription initiation factor (TIF), TIF-IE, is required for homogeneous Acanthamoeba castellanii TIF-IB (SL1) to form a committed complex.

The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity from Acanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460, 000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 +/- 3 pM. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promoter-containing DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex.

Acanthamoeba↗
PubMed1997

The fundamental ribosomal RNA transcription initiation factor-IB (TIF-IB, SL1, factor D) binds to the rRNA core promoter primarily by minor groove contacts.

Acanthamoeba castellanii transcription initiation factor-IB (TIF-IB) is the TATA-binding protein-containing transcription factor that binds the rRNA promoter to form the committed complex. Minor groove-specific drugs inhibit TIF-IB binding, with higher concentrations needed to disrupt preformed complexes because of drug exclusion by bound TIF-IB. TIF-IB/DNA interactions were mapped by hydroxyl radical and uranyl nitrate footprinting. TIF-IB contacts four minor grooves in its binding site. TIF-IB and DNA wrap around each other in a right-handed superhelix of high pitch, so the upstream and downstream contacts are on opposite faces of the helix. Dimethyl sulfate protection assays revealed limited contact with a few guanines in the major groove. This detailed analysis suggests significant DNA conformation dependence of the interaction.

Base Sequence↗
PubMed1997

Identification of previously unrecognized common elements in eukaryotic promoters. A ribosomal RNA gene initiator element for RNA polymerase I.

A new ribosomal RNA promoter element with a functional role similar to the RNA polymerase II initiator (Inr) was identified. This sequence, which we dub the ribosomal Inr (rInr) is unusually conserved, even in normally divergent RNA polymerase I promoters. It functions in the recruitment of the fundamental, TATA-binding protein (TBP)-containing transcription factor, TIF-IB. All upstream elements of the exceptionally strong Acanthamoeba castellanii ribosomal RNA core promoter, to within 6 base pairs of the transcription initiation site (tis), can be deleted without loss of specific transcription initiation. Thus, the A. castellanii promoter can function in a manner similar to RNA polymerase II TATA-less promoters. Sequence-specific photo-cross-linking localizes a 96-kDa subunit of TIF-IB and the second largest RNA polymerase I subunit (A133) to the rInr sequence. A185 also photo-cross-links when polymerase is stalled at +7.

Acanthamoeba↗
PubMed1995

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

Acanthamoeba↗
PubMed1995

Coordinate regulation of ribosomal component synthesis in Acanthamoeba castellanii: 5S RNA transcription is down regulated during encystment by alteration of TFIIIA activity.

Transcription of large rRNA precursor and 5S RNA were examined during encystment of Acanthamoeba castellanii. Both transcription units are down regulated almost coordinately during this process, though 5S RNA transcription is not as completely shut down as rRNA transcription. The protein components necessary for transcription of 5S RNA and tRNA were determined, and fractions containing transcription factors comparable to TFIIIA, TFIIIB, and TFIIIC, as well as RNA polymerase III and a 3'-end processing activity, were identified. Regulation of 5S RNA transcription could be recapitulated in vitro, and the activities of the required components were compared. In contrast to regulation of precursor rRNA, there is no apparent change during encystment in the activity of the polymerase dedicated to 5S RNA expression. Similarly, the transcriptional and promoter-binding activities of TFIIIC are not altered in parallel with 5S RNA regulation. TFIIIB transcriptional activity is unaltered in encysting cells. In contrast, both the transcriptional and DNA-binding activities of TFIIIA are strongly reduced in nuclear extracts from transcriptionally inactive cells. These results were analyzed in terms of mechanisms for coordinate regulation of rRNA and 5S RNA expression.

Acanthamoeba↗
PubMed1995

Site-directed photo-cross-linking of rRNA transcription initiation complexes.

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

Acanthamoeba↗
PubMed1994

Sequence organization of the Acanthamoeba rRNA intergenic spacer: identification of transcriptional enhancers.

The primary sequence of the entire 2330 bp intergenic spacer of the A.castellanii ribosomal RNA gene was determined. Repeated sequence elements averaging 140 bp were identified and found to bind a protein required for optimum initiation at the core promoter. These repeated elements were shown to stimulate rRNA transcription by RNA polymerase I in vitro. The repeats inhibited transcription when placed in trans, and stimulated transcription when in cis, in either orientation, but only when upstream of the core promoter. Thus, these repeated elements have characteristics similar to polymerase I enhancers found in higher eukaryotes. The number of rRNA repeats in Acanthamoeba cells was determined to be 24 per haploid genome, the lowest number so far identified in any eukaryote. However, because Acanthamoeba is polyploid, each cell contains approximately 600 rRNA genes.

Acanthamoeba↗
PubMed1994

TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.

Acanthamoeba↗
PubMed1993

Promoter opening (melting) and transcription initiation by RNA polymerase I requires neither nucleotide beta,gamma hydrolysis nor protein phosphorylation.

With some bacterial RNA polymerases and in eukaryotic RNA polymerase II, DNA melting during initiation requires the coupling of energy derived from beta,gamma hydrolysis of ATP. A detailed analysis of this possible requirement for eukaryotic RNA polymerase I reveals no such requirement. However, in some cases, beta,gamma non-hydrolyzable derivatives (beta,gamma imido or methylene) of nucleotide substrates have been found to significantly inhibit transcription initiation because of their inefficient use as the first nucleotide of the transcript. In addition, the results presented here show that protein kinase activity is not required as an integral part of transcription initiation by RNA polymerase I. Prior phosphorylation of proteins participating in the process is not ruled out.

Acanthamoeba↗
PubMed1993

Binding of the RNA polymerase I transcription complex to its promoter can modify positioning of downstream nucleosomes assembled in vitro.

We have studied the reconstitution of chromatin-like structures in vitro, using purified RNA polymerase I transcription complexes and histone octamers. The plasmid construct used in these studies is a pUC8 derivative in which we have inserted an RNA polymerase I core promoter region of Acanthamoeba castellanii upstream of four repeats of the 5 S rDNA nucleosome positioning sequence (208 base pairs) from Lytechinus variegatus. When histone octamers were reconstituted onto the naked DNA template, the expected nucleosome positioning previously observed using tandem repeats of the same 208-base pair fragment was not obtained (as assayed by restriction enzyme digestion mapping of the inserted region of the plasmid). We show that the location of the RNA polymerase I core promoter region with regard to the tandemly repeated 208-base pair positioning sequence is a major determinant in the positioning of the histone octamers. Reconstituting first with the stalled transcription complex excluded octamers from the promoter region and recovered the expected nucleosome positioning downstream on the four repeats of the 5 S positioning sequence. The observed competition between histone octamers and the transcription complex for the promoter region suggests a great similarity with what has been reported from in vitro studies of RNA polymerase II and III transcription systems. We may be looking at a mechanism of regulation of transcription for the RNA polymerase I.

Acanthamoeba↗
PubMed1992

An exonuclease requiring an intact helical stem for specificity produces the 3' end of Acanthamoeba castellanii 5 S RNA.

A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.

Acanthamoeba↗
PubMed1992

Purification of components required for accurate transcription of ribosomal RNA from Acanthamoeba castellanii.

The components required for specific transcription of ribosomal RNA were isolated from logarithmically growing Acanthamoeba castellanii. The transcription initiation factor fraction, TIF, and RNA polymerase I were extracted from whole cells at 0.35 M KCl. The extract was fractionated with polyethylenimine, then chromatographed on phosphocellulose (P11) which resulted in the separation of TIF from RNA polymerase I. The fractions containing TIF were further chromatographed on DEAE cellulose (DE52), Heparin Affigel, and Matrex green agarose, followed by sedimentation through glycerol gradients. TIF was purified approximately 17,000-fold, and shown to have a native molecular weight of 289 kD, and to bind specifically to rRNA promoter sequences by DNase I footprinting. The addition of homogeneous RNA polymerase I to this complex permitted the initiation of specific transcription in vitro. The phosphocellulose fractions containing RNA polymerase I were chromatographed on DEAE cellulose, Heparin-Sepharose, DEAE-Sephadex, and sedimented through sucrose gradients. Polymerase I was purified to apparent homogeneity with a yield of 8.1% and a specific activity of 315. It contained one fewer subunit than previously reported. DNase I protection experiments demonstrated that in both partially purified and homogeneous fractions, RNA polymerase I was capable of stable binding to the TIF-rDNA complex, and correctly initiating transcription on rDNA templates.

Acanthamoeba↗