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.

The association of TIF-IA and polymerase I mediates promoter recruitment and regulation of ribosomal RNA transcription in Acanthamoeba castellanii.

Large amounts of energy are expended for the construction of the ribosome during both transcription and processing, so it is of utmost importance for the cell to efficiently regulate ribosome production. Understanding how this regulation occurs will provide important insights into cellular growth control and into the coordination of gene expression mediated by all three transcription systems. Ribosomal RNA (rRNA) transcription rates closely parallel the need for protein synthesis; as a cell approaches stationary phase or encounters conditions that negatively affect either growth rate or protein synthesis, rRNA transcription is decreased. In eukaryotes, the interaction of RNA polymerase I (pol I) with the essential transcription initiation factor IA (TIF-IA) has been implicated in this downregulation of transcription. In agreement with the first observation that rRNA transcription is regulated by altering recruitment of pol I to the promoter in Acanthamoeba castellanii, we show here that pol I and an 80-kDa homologue of TIF-IA are found tightly associated in pol I fractions competent for specific transcription. Disruption of the pol I-TIF-IA complex is mediated by a specific dephosphorylation of either pol I or TIF-IA. Phosphatase treatment of TIF-IA-containing A. castellanii pol I fractions results in a downregulation of both transcriptional activity and promoter binding, reminiscent of the inactive pol I fractions purified from encysted cells. The fraction of pol I competent for promoter recruitment is enriched in TIF-IA relative to that not bound by immobilized promoter DNA. This downregulation coincides with an altered electrophoretic mobility of TIF-IA, suggesting at least it is phosphorylated.

Photocross-linking of the RNA polymerase I preinitiation and immediate postinitiation complexes: implications for promoter recruitment.

The architecture of eukaryotic rRNA transcription complexes was analyzed, revealing facts significant to the RNA polymerase (pol) I initiation process. Functional initiation and elongation complexes were mapped by site-specific photocross-linking to template DNA. Polymerase I is recruited to the promoter via protein-protein interactions with DNA-bound transcription initiation factor-IB. The latter's TATA-binding protein (TBP) and TAFs photocross-link to the promoter from -78 to +10 relative to the tis (+1). Although TBP does not bind DNA using its TATA-binding saddle, it does photocross-link to a 22-bp sequence that does not resemble a TATA box. Only TAF(I)96 (the mammalian TAF(I) 68, yeast Rrn7p homolog) overlaps significantly with the DNA interaction cleft of pol I based on modeling to the pol II crystal structure. None of the pol I-specific subunits that are localized on the lips of the cleft (A49 and A34.5) or the pol I-specific stalk (A43 and A14) cross-link to DNA. Pol I does not extend significantly upstream of the promoter-proximal border of the factor complex (-11 to -14), and similarly in the promoter proximal elongation complex, the enzyme does not contact DNA upstream of its normal exit from the cleft.

Purification and characterization of transcription factor IIIA from Acanthamoeba castellanii.

TFIIIA is required to activate RNA polymerase III transcription from 5S RNA genes. Although all known TFIIIA homologs harbor nine zinc fingers that mediate DNA binding, very limited sequence homology is found among these proteins, which reflects unique properties of some TFIIIA homologs. For example, the Acanthamoeba castellanii homolog directly regulates 5S RNA transcription. We have purified and characterized A.castellanii TFIIIA (AcTFIIIA) as a step toward obtaining a clearer understanding of these differences and of the regulatory process. AcTFIIIA is 59 kDa, significantly larger than all other TFIIIA homologs isolated to date. Nevertheless, it exhibits a DNase I footprint very similar to those produced by the smaller vertebrate TFIIIA homologs, but distinct from the smaller footprint of the 51 kDa TFIIIA from Saccharomyces cerevisiae. Similar footprinting is not reflected in greater sequence similarity between the A.castellanii and vertebrate promoters.

A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.