RESUMO
The structures of the reaction products are the basis for novel polymerase assays using the atomic force microscope (AFM). Polymerases are the enzymes involved in transcription and replication of DNA. Rapid semiquantitative estimates of the activity of DNA polymerases such as Sequenase, Taq polymerase, and AMV reverse transcriptase and RNA polymerases (RNAP) such as Escherichia coli RNAP were obtained from AFM images of the nucleic acids after polymerase reactions. DNA polymerases were assayed via replication of the single-stranded φX-174 virion. RNAP was assayed via transcription, using a rolling circle DNA template that produces long strands of RNA. In some cases, AFM was better than agarose gel electrophoresis for assaying DNA polymerase activity, since aggregation prevented the DNA from entering the agarose gel. Extended molecules of single-stranded RNA synthesized with the rolling circle DNA template showed varied conformations and degrees of stretching. Some structural differences were observed between two RNAs-a ribozyme concatamer and an RNA with 90% purines.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/biossíntese , RNA/biossíntese , Bacteriófago phi X 174/genética , DNA/ultraestrutura , DNA Viral/metabolismo , Eletroforese em Gel de Ágar , Escherichia coli/enzimologia , Microscopia de Força Atômica/métodos , RNA/ultraestrutura , DNA Polimerase Dirigida por RNA/metabolismo , Taq Polimerase/metabolismo , Transcrição GênicaAssuntos
DNA Circular/genética , DNA de Cadeia Simples/genética , RNA/genética , Sequência de Bases , DNA Circular/síntese química , DNA de Cadeia Simples/síntese química , RNA Polimerases Dirigidas por DNA , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , RNA/química , RNA Catalítico/química , RNA Catalítico/genética , Moldes Genéticos , Transcrição GênicaRESUMO
Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports. We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro. The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters. Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (approximately 7,500 nt). During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length. Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans. In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans. This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.
