Marvin R. Paule

Promoter occlusion during ribosomal RNA transcription

The ability of RNA polymerase I to read through a promoter-bound transcription initiation factor (TIF) was assessed using a dimeric ribosomal RNA gene promoter. Transcription from the upstream promoter is unaffected by TIF bound to the downstream promoter; RNA polymerase I is freely able to read through DNA-bound TIF. In contrast, transcription from the downstream promoter is inhibited by the passage of RNA polymerase I. Readthrough by RNA polymerase I disrupts the downstream TIF-DNA complex, and alters the TIF footprint. A general function for sequences leading to transcription termination upstream of rRNA or other promoters may be the prevention of promoter occlusion in tandem arrays of genes.

Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification

Forms of RNA polymerase I prepared from growing or encysted Acanthamoeba are equal in the ability to transcribe poly(dl:dC). Polymerase from cysts, whose rRNA genes are transcriptionally inactive, is unable to utilize the rDNA promoter in vitro, whereas the transcription initiation factor from cysts is fully able to bind the promoter and direct transcription. Footprinting shows that polymerase from cysts is functionally inactive because of its inability to bind to the promoter. The polymerase footprint moves downstream the appropriate number of base pairs upon various nucleotide additions, without affecting the factor footprint. These results support our hypothesis that rRNA synthesis in eukaryotes is regulated by polymerase I modification and not by alterations to additional DNA-binding proteins.

Comparative subunit composition of the eukaryotic nuclear RNA polymerases

Abstract Multiple eukaryotic RNA polymerases have enormously complex subunit compositions. However, comparison studies reveal that these polypeptides fall into several categories which may reflect their functional role in gene transcription.

Eukaryotic RNA polymerase I promoter binding is directed by protein contacts with transcription initiation factor and is DNA sequence-independent.

RNA polymerase I binding to the eukaryotic ribosomal RNA gene promoter-transcription initiation factor (TIF) complex was examined by in vitro transcription and footprinting of a series of spacer mutants. Polymerase binds efficiently to the TIF-promoter complex independently of the DNA sequence in the polymerase interaction region and initiates transcription a fixed distance downstream of the TIF binding site on AT-rich templates. Methidiumpropyl-EDTA.FE(II) footprinting confirms minimal contacts between polymerase and DNA. We infer that polymerase is directed to the promoter by a DNA sequence-independent mechanism, solely by protein-protein contacts with TIF. An initiation step subsequent to binding requires special sequence characteristics in the transcription start site region.

Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decreased specific RNA synthesis in vitro. These were within the region which is protected from DNase I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

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).

DNA-dependent RNA polymerases from Acanthamoeba castellanii: Properties and levels of activity during encystment

Three DNA-dependent RNA polymerases have been isolated and partially purified from trophozoites of Acanthamoeba castellanii. Separated by DEAE-Sephadex chromatography, they have been designated polymerases, I, IIa and IIB according to their alpha-amanitin sensitivity and kinetic properties. I is completely insensitive to alpha-amanitin. IIa and IIb are sensitive to low concentrations (0.1 mug/ml) of alpha-amanitin; however, in order to achieve 100% inhibition much higher concentrations (130 mug/ml) are needed. Both I and II (a or b) have rather broad ionic strength optima (0.06--0.10 M (NH4)2SO4). All three prefer denatured over native DNA (I, 4:1; II, 2:1). Polymerase I utilizes magnesium better than manganese as divalent cation whereas II prefers manganese. When Acanthamoeba is transferred to a medium lacking nutrients, the cells undergo a synchronous differentiation resulting in cyst formation. In general agreement with the decrease in the rate of synthesis of its product (rRNA), the amount of polymerase I decreases relative to the amanitin sensitive polymerase(s). However, the absolute amount of polymerase I does not change. Rather, the levels of the amanitin sensitive enzymes increase during the first 10 h of encystment. Since the overall RNA synthesis rate decreases, these results suggest that the transcription rate is not controlled by specific enzyme levels alone.

The Use of Diethyl Pyrocarbonate and Potassium Permanganate as Probes for Strand Separation and Structural Distortions in DNA

Diethyl pyrocarbonate (DEPC) and potassium permanganate are useful reagents for detecting DNA distortions, especially melted regions. Unlike most other footprinting methods, these reagents can detect such distortions even within the regions of protein-DNA complexes normally protected in other footprinting techniques. Further, reactions are very robust, so that distorted regions can be detected even under conditions where efficiency of DNA-protein complex formation is not high. DEPC reacts with bases that are fully or partially unstacked in DNA, in the preferential order adenosine > guanine >> cytosine. Permanganate reacts strongly with thymine in unstacked regions of DNA, and exhibits only very weak reaction with guanine, cytosine, or adenine. The combination of both reagents gives excellent coverage of all sequence regions of DNA. Because reaction requires unstacking, the two reagents detect both melted regions and regions that are unstacked because of other distortions such as bending. Permanganate has the additional advantage that it can be utilized in living cells.

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.

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

Abstract. 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.