RESUMO
We have generated 14 recombinant RNA templates for Q beta replicase, each having either an exogenous inverted repeat sequence or a sequence with no repeat. These templates were used to initiate in vitro replication by Q beta replicase in amounts that saturated the enzyme. We observed that replication rates for RNAs that putatively contained secondary structures in the recombinant sequences ranged from 33 to 69% that of a wild-type MDV-1 RNA control, regardless of the size of the inserted hairpin. Moreover, most of the newly synthesized RNA was present as single strands. Alternatively, RNAs that contained exogenous sequences not expected to form secondary structures exhibited replication rates less than 25% that of MDV-1. In each case, the reaction rate was correlated with the length of the insertion, and the majority of product RNA consisted of duplexed molecules (complementary plus and minus strands hybridized together). When these same recombinant RNAs were used in reactions in which the molar amount of RNA template was 10(6)-10(7) times lower than that of the replicase, only those that putatively contained secondary structures survived in the replication reaction. Our results are consistent with the theory that hairpin structure formation during RNA synthesis by Q beta replicase directly influences the regeneration of single-stranded RNA products.
Assuntos
Colífagos/crescimento & desenvolvimento , Q beta Replicase/metabolismo , RNA Viral/metabolismo , Replicação Viral , Sequência de Bases , Clonagem Molecular , DNA/genética , HIV/genética , Ligação de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular , RNA Viral/ultraestrutura , Mapeamento por RestriçãoRESUMO
RNA synthesis by T7 RNA polymerase or SP6 RNA polymerase is 100-1000 times more sensitive to the presence of the 3'-deoxyribonucleoside 5'-triphosphate chain terminators than is RNA synthesis by Escherichia coli RNA polymerase or Q beta replicase. These ribonucleotide analogues do not alter the specificity of each polymerase for its own promoters nor do they alter the site at which synthesis is initiated. Transcription by T7 RNA polymerase or SP6 RNA polymerase in the presence of relatively low concentrations of these chain terminators offers a useful route for determining the nucleotide sequence of any DNA segment that is inserted immediately downstream from a homologous bacteriophage promoter. This sequencing procedure was used to explore the effects that different dinucleotides have on the specificity of initiation at two different T7 RNA polymerase promoters.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Regiões Promotoras Genéticas , Fagos T/enzimologia , Transcrição Gênica , Sequência de Bases , Enzimas de Restrição do DNA , DNA Viral/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Escherichia coli/genética , Plasmídeos , Q beta Replicase/metabolismo , Fagos T/genéticaRESUMO
Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described.
