Enzymatic synthesis of 2′-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates (original) (raw)
Related papers
Nucleic Acids Research, 1999
A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2′-group, facilitates incorporation of non-canonical substrates into nucleic acids. However, transcripts containing a high percentage of non-canonical NMPs are poorly extended due to effects of the 2′-substituents on the transcript:template hybrid conformation. We tested the addition of compounds that stabilize A-type helix geometry to the reaction. High concentrations of polyamines, together with other changes in reaction conditions, greatly increased the synthesis of transcripts heavily substituted with non-canonical ribose 2′-groups. Template structures that facilitate promoter opening increased the efficiency of reactions where non-canonical substrates were incorporated during transcription of +1 to +6.
A mutant T7 RNA polymerase as a DNA polymerase
The EMBO Journal, 1995
We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non-conservative mutations of this tyrosine also reduce discrimination between deoxyriboand ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction.
On the early evolution of RNA polymerase
Journal of Molecular Evolution, 1988
The lines of evidence suggesting that RNA preceded double-stranded DNA as an informational macromolecule are briefly reviewed. RNA polymerase is hypothesized to have been one of the earliest proteins to appear. It is argued that an important vestige of the original enzyme is found in the contemporary eubacterial β′ subunit of DNA-dependent RNA polymerase and its homologues among the archaebacterial and eukaryotic enzymes. The evidence that supports a catalytic role in replicase activity of this polypeptide is reviewed. It is suggested that several characteristics of theEscherichia coli transcriptional apparatus are relatively recent evolutionary developments. The phylogenetic importance of the eubacterial β′ subunit from RNA polymerase and its homologues is emphasized, because it allows the study of the evolutionary relationships of the major cellular lines (eubacteria, archaebacteria, and eukaryotes) as well as of some viral lineages.
Initiation by the DNA-dependent RNA polymerase
Proceedings of the National Academy of Sciences, 1966
This communication presents evidence which indicates that RNA synthesis by the DNA-dependent RNA polymerase occurs in three steps: (1) Association: DNA + Enzyme = DNA-enzyme. (2) Initiation: DNA-enzyme + purine nucleotide > [DNA-enzyme-purine1 L nucleotide I (3) Polymerization: [DNA-enzyme-purine] + NTP DNA-enzyme-oligoribonucleotide + PP1. The formation of the DNA-enzyme complex, step 1, can be inhibited by high ionic strength. When initiation occurs, a different DNA-enzyme complex is formed in the presence of purine nucleoside triphosphates which is not as easily dissociated by high ionic strength. The initiation complex can also be detected by a specially devised membrane assay. The exact nature of this complex is not established, but for its formation, a relatively high level of purine nucleotide is required. It will be shown that the association DNA-enzyme complex differs from the initiation DNAenzyme complex. The process of initiation is rate-limiting at low nucleoside triphosphate concentrations and purine nucleotides in relatively high concentrations overcome this limitation. In step 3, there is no differential effect of purine over pyrimidine nucleotides. The effect of purine nucleotides on initiation correlates with the observations of Maitra and Hurwitz' that the purine nucleoside triphosphates are the predominant 5'-terminal nucleotides found in RNA synthesized in vitro.
Proceedings of The National Academy of Sciences, 1997
The traditional classification of nucleic acid polymerases as either DNA or RNA polymerases is based, in large part, on their fundamental preference for the incorporation of either deoxyribonucleotides or ribonucleotides during chain elongation. The refined structure determination of Moloney murine leukemia virus reverse transcriptase, a strict DNA polymerase, recently allowed the prediction that a single amino acid residue at the active site might be responsible for the discrimination against the 2OH group of an incoming ribonucleotide. Mutation of this residue resulted in a variant enzyme now capable of acting as an RNA polymerase. In marked contrast to the wild-type enzyme, the K m of the mutant enzyme for ribonucleotides was comparable to that for deoxyribonucleotides. The results are consistent with proposals of a common evolutionary origin for both classes of enzymes and support models of a common mechanism of nucleic acid synthesis underlying catalysis by all such polymerases.
Structure and evolution of prokaryotic and eukaryotic RNA polymerases: a model
Journal of Theoretical Biology, 1987
A comparative overview of the subunit taxonomy and sequences of eukaryotic and prokaryotic RNA polymerases indicates the presence of a core structure conserved between both sets of enzymes. The differentiation between prokaryotic and eukaryotic polymerases is ascribed to domains and subunits peripheral to the largely conserved central structure. Possible subunit and domain functions are outlined. The core's flexible shape is largely determined by the elongated architecture of the two largest subunits, which can be oriented along the DNA axis with their bulkier amino-terminal head regions looking towards the 3' end of the gene to be transcribed and their more slender carboxyl-terminal domains at the tail end of the enzyme. The two largest prokaryotic subunits appear originally derived from a single gene.
Origin and Evolution of RNA-Dependent RNA Polymerase
Frontiers in Genetics
RNA-dependent RNA polymerases (RdRp) are very ancient enzymes and are essential for all viruses with RNA genomes. We reconstruct the origin and evolution of this polymerase since the initial stages of the origin of life. The origin of the RdRp was traced back from tRNA ancestors. At the origin of the RdRp the most ancient part of the protein is the cofactor-binding site that had the capacity of binding to simple molecules as magnesium, calcium, and ribonucleotides. Our results suggest that RdRp originated from junctions of proto-tRNAs that worked as the first genes at the emergence of the primitive translation system, where the RNA was the informational molecule. The initial domain, worked as a building block for the emergence of the fingers and thumb domains. From the ancestral RdRp, we could establish the evolutionary stages of viral evolution from a rooted ancestor to modern viruses. It was observed that the selective pressure under the RdRp was the organization and functioning of the genome, where RNA double-stranded and RNA single-stranded virus formed a separate group. We propose an evolutionary route to the polymerases and the results suggest an ancient scenario for the origin of RNA viruses.