Daniel F. Bogenhagen

Purified RNA polymerase III accurately and efficiently terminates transcription of 5s RNA genes

RNA polymerase III was purified to about 90% homogeneity from ovarian tissue of Xenopus laevis. The enzyme accurately initiates and terminates transcription of 5S RNA synthesis in isolated nuclei, but not when naked 5S DNA is used as a template. A sensitive hybridization technique was used to demonstrate that the purified polymerase, even when supplemented with a transcription factor that binds specifically to the 5S RNA gene internal control region, is unable to initiate synthesis at the start site of the 5S RNA gene. However, the polymerase alone terminates transcription at precisely the same site that is recognized in vivo and in complete transcription extracts. The purified polymerase distinguishes between weak and strong terminator sequences with the same relative efficiency as the enzyme in complete extracts. We conclude that the pure enzyme can recognize the simple consensus sequence found at the end of genes transcribed by RNA polymerase III.

Repair of a synthetic abasic site in DNA in a Xenopus laevis oocyte extract.

Covalently closed circular DNA containing a synthetic analog of an abasic site at a unique position was used as a substrate to study DNA repair. Incubation of this DNA in Xenopus laevis oocyte extracts resulted in rapid cleavage of the DNA at the abasic site by a class II apurinic-apyrimidinic endonuclease, followed by complete repair within 40 min. Nicked circular DNAs persisted for several minutes before repair by an ATP-dependent DNA synthesis reaction. The repair-related DNA synthesis was localized within 3 or 4 nucleotides surrounding the abasic site. These results are consistent with the short-patch repair reported for DNA damage at heterogeneous sites in human cells (J. D. Regan and R. B. Setlow, Cancer Res. 34:3318-3325, 1974).

Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription.

The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor eluted from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA(Phe) and the displacement loop.

TFIIIA binds to different domains of 5S RNA and the Xenopus borealis 5S RNA gene.

We have established the conditions for the reassociation of 5S RNA and TFIIIA to form 7S particles. We tested the ability of altered 5S RNAs to bind TFIIIA, taking advantage of the slower mobility of 7S particles compared with free 5S RNA in native polyacrylamide gels. Linker substitution mutants were constructed encompassing the entire gene, including the intragenic control region. In vitro transcripts of the linker substitution mutants were tested for their ability to bind TFIIIA to form 7S ribonucleoprotein particles. Altered 5S RNAs with base changes in or around helices IV and V, which would interfere with the normal base pairing of that region, showed decreased ability to bind TFIIIA. The transcripts of some mutant genes that were efficiently transcribed (greater than 50% of wild-type efficiency) failed to bind TFIIIA in this gel assay. In contrast, the RNA synthesized from a poorly transcribed mutant, LS 86/97, in which residues 87 to 96 of the RNA were replaced in the single-stranded loop at the base of helix V, bound TFIIIA well. The data indicate that TFIIIA binds to different domains in the 5S RNA gene and 5S RNA.

Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.

Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIIIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIIIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic 5S RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA.

The 165-kDa DNA topoisomerase I from Xenopus laevis oocytes is a tissue-specific variant

Two forms of topoisomerase I can be purified from Xenopus laevis. A protein with a molecular mass of 165 kDa has been identified as topoisomerase I in ovaries (Richard and Bogenhagen, 1989. J. Biol. Chem. 264, 4704-4709). When a similar purification is performed using liver tissue, topoisomerase I is purified as a 110-kDa protein. Separate rabbit antisera were raised against oocyte and liver topoisomerase I polypeptides. Each antiserum reacts in immunoblotting or immunoprecipitation procedures only with the tissue-specific topoisomerase I polypeptide against which it was generated. The failure of the antiserum raised against liver topoisomerase I to cross-react with the oocyte enzyme suggests that the smaller topoisomerase I is not derived from the 165-kDa oocyte enzyme by proteolysis. X. laevis tissue culture cells lysed and processed in the presence of SDS contain the 110-kDa form of topoisomerase I. The 165-kDa form of topoisomerase I disappears during oocyte maturation in vitro.