Architecture of protein and DNA contacts within the TFIIIB-DNA complex

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.     

The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases

We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum. The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases. Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized. Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin. Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities. In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak. A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C. However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus. In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A. These findings suggest that both enzymes from P. occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.     

The hyperthermophilic bacterium Thermotoga maritima has two different classes of family C DNA polymerases: evolutionary implications

Bacterial DNA polymerase III (family C DNA polymerase), the principal chromosomal replicative enzyme, is known to occur in at least three distinct forms which have provisionally been classified as class I ( Escherichia coli DNA pol C-type), class II ( Bacillus subtilis DNA pol C-type) and class III (cyanobacteria DNA pol C-type). We have identified two family C DNA polymerase sequences in the hyperthermophilic bacterium Thermotoga maritima. One DNA polymerase consisting of 842 amino acid residues and having a molecular weight of 97 213 belongs to class I. The other one, consisting of 1367 amino acid residues and having a molecular weight of 155 361, is a member of class II. Comparative sequence analyses suggest that the class II DNA polymerase is the principal DNA replicative enzyme of the microbe and that the class I DNA polymerase may be functionally inactive. A phylogenetic analysis using the class II enzyme indicates that T.maritima is closely related to the low G+C Gram-positive bacteria, in particular to Clostridium acetobutylicum, and mycoplasmas. These results are in conflict with 16S rRNA-based phylogenies, which placed T.maritima as one of the deepest branches of the bacterial tree.