David L. Thurlow

Unidentified open reading frames in the genome of Methanococcus jannaschii are similar in sequence to an archaebacterial gene for tRNA nucleotidyltransferase.

Abstract. The protein sequence of ATP/CTP:tRNA nucleotidyltransferase (cca) from Sulfolobus shibatae was used to search open reading frames in the genome of Methanococcus jannaschii. Translations of two unidentified open reading frames showed significant sequence similarity to portions of the Sulfolobus cca protein. When the two open reading frames were joined together, the expanded open reading frame was similar in sequence to the entire Sulfolobus cca protein and displayed features of the active site signature sequence proposed for members of class I enzymes within the superfamily of nucleotidyltransferases (Yue et al., 1996, RNA 2, 895–908). A possible UUG start codon was identified based on significant sequence similarity of the resulting amino-terminal region to that of Sulfolobus, and on a six-base complementarity between an adjacent upstream sequence and Methanococcus 16S rRNA.

Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase–helicase from Arabidopsis thaliana

An open reading frame from Arabidopsisthaliana, which is highly homologous to the human mitochondrial DNA helicase TWINKLE, was previously cloned, expressed, and shown to have DNA primase and DNA helicase activity. The level of DNA primase activity of this Arabidopsis Twinkle homolog (ATH) was low, perhaps due to an incomplete zinc binding domain (ZBD). In this study, N-terminal truncations of ATH implicate residues 80–102 interact with the RNA polymerase domain (RPD). In addition, chimeric proteins, constructed using domains from ATH and the well-characterized T7 phage DNA primase–helicase gp4, were created to determine if the weak primase activity of ATH could be enhanced. Two chimeric proteins were constructed: ATHT7 contains the ZBD and RPD domains of ATH tethered to the helicase domain of T7, while T7ATH contains the ZBD and RPD domains of T7 tethered to the helicase domain of ATH. Both chimeric proteins were successfully expressed and purified in E. coli, and assayed for traditional primase and helicase activities. T7ATH was able to generate short oligoribonucleotide primers, but these primers could not be cooperatively extended by a DNA polymerase. Although T7ATH contains the ATH helicase domain, it exhibited few of the characteristics of a functional helicase. ATHT7 lacked primase activity altogether and also demonstrated only weak helicase activities. This work demonstrates the importance of interactions between structurally and functionally distinct domains, especially in recombinant, chimeric proteins.

Fold recognition, homology modeling, docking simulations, kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii

ATP/CTP:tRNA nucleotidyltransferases (NTases) and poly(A) polymerases (PAPs) belong to the same superfamily and their catalytic domains are remotely related. Based on the results of fold‐recognition analysis and comparison of secondary structure patterns, we predicted that these two NTase families share three domains, corresponding to “palm,” “fingers,” and “fingernails” in the PAP crystal structure. A homology model of tRNA NTase from Methanococcus jannaschii was constructed. Energy minimization calculations of enzyme‐nucleotide complexes and computer‐aided docking of nucleotides onto the enzymes surface were carried out to explore possible ATP and CTP binding sites. Theoretical models were used to guide experimental analysis. Recombinant His‐tagged enzyme was expressed in Escherichia coli, and kinetic properties were characterized. The apparent KM for CTP was determined to be 38 μM, and the apparent KM for ATP was 21 μM. Three mutations of basic amino acids to alanine were created in a highly conserved region predicted to be in the vicinity of the nucleotide binding site. A deletion was also constructed to remove the C‐terminal structural domain defined by the model; it retained about 1% of wild type enzymatic activity using CTP as co‐substrate, confirming that detectable catalytic activity is exhibited by the N‐terminal domain, as defined by the model. Our results suggest a mechanism of differential ATP and CTP binding, which explains how the tRNA NTase, having only one catalytic site, utilizes different nucleotide triphosphates depending on the nature of the tRNA substrate. Proteins 2003;51:349–359.