Crucial role of the RNA:DNA hybrid in the processivity of transcription.
We present an approach for studying the role of complementary nucleic acid interactions in transcription elongation by E. coli RNA polymerase (RNAP). The method involves in vitro reconstitution of a catalytically active elongation complex (EC) by the addition of RNAP to a single-strand DNA oligonucleotide containing the preannealed RNA primer, followed by incorporation of the complementary nontemplate DNA oligonucleotide. In all parameters tested, the reconstituted complex is indistinguishable from normal EC obtained by promoter-specific initiation. Using RNA primers of different lengths, which were fully or partially complementary to the DNA, we determined the minimal transcript length and the degree of its template pairing that is required to stabilize protein/ nucleic acid interactions in EC to the high level characteristic of normal transcription. Our data show that a hybrid at least 9 nt long, formed between the template DNA and 3'-proximal RNA transcript, is necessary for the high processivity of EC during RNA chain elongation.
ABL-Basic Research Program, National Cancer Institute-Frederic Cancer Research and Development Center, Maryland 21702-1201, USA.
I
I
Sidorenkov
N
N
Komissarova
M
M
Kashlev
D001426:Bacterial Proteins / Q000378:metabolism*; D001482:Base Composition; D002384:Catalysis; D002474:Cell-Free System; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D046911:Macromolecular Substances; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012318:RNA Polymerase I / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000502:physiology*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; C034477:RNA primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D012318:RNA Polymerase I