RESUMO
A polynucleotide kinase from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus was identified, expressed, and purified. This polynucleotide kinase was demonstrated to have a 5'-kinase domain as well as a 3'-phosphohydrolase domain. The RM378 polynucleotide kinase had limited sequence similarity to the 5'-kinase domain of the T4 bacteriophage polynucleotide kinase, but apparent homology was not evident within the 3'-phosphohydrolase domain. The domain order of RM378 polynucleotide kinase was reversed relative to that of the T4 polynucleotide kinase. The RM378 phosphohydrolase domain displayed some sequence similarity with the bacterial poly(A) polymerase family, including an HD motif characteristic of the diverse superfamily of metal-dependent HD phosphohydrolases. The RM378 polynucleotide kinase was biochemically characterized and shown to possess 5'-kinase activity on RNA and single- and double-stranded DNA at elevated temperatures. It also showed phosphohydrolase activity on 2':3'-cyclic adenosine monophosphate. This description of the RM378 polynucleotide kinase, along with the recently described RM378 RNA ligase, suggests that the RM378 bacteriophage has to counter a similar anti-phage mechanism in R. marinus as the one that the T4 phage has to counter in Escherichia coli.
Assuntos
Bacteriófagos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/química , Polinucleotídeo 5'-Hidroxiquinase/química , Estrutura Terciária de ProteínaRESUMO
Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.