Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription.
Ternary complexes of RNA polymerase, bearing the nascent RNA transcript, are intermediates in the synthesis of all RNAs and are regulatory targets of factors that control RNA chain elongation and termination. To study the catalytic and regulatory properties of RNA polymerases during elongation, we have developed methods for the preparation of these intermediates halted at defined positions along a DNA template. To our surprise, some of these halted complexes undergo a reaction in which the RNA transcript is cleaved up to 10 nucleotides from its 3'-terminal growing point. The 5'-terminal fragment, bearing a free 3'-OH residue, remains bound to the RNA polymerase-DNA complex and can resume elongation, whereas the 3'-terminal oligonucleotide of 2-10 nucleotides, bearing a 5'-phosphate, is released. RNA cleavage occurs only in the ternary complex and requires a divalent metal ion such as Mg2+. Since RNA polymerases are believed to have a single catalytic site for nucleotide addition, this reaction is unlikely to be due to hydrolysis catalyzed by this site comparable to the 3'----5' exonuclease activity associated with the catalytic center found for some DNA polymerases. Nor is this reaction easily explained by models for transcription elongation that postulate a 12-base-pair DNA.RNA hybrid as intermediate. Instead, we suggest that this is an unusual kind of protein-facilitated reaction in which tight binding of the RNA product to the enzyme strains the RNA phosphodiester linkage, resulting in cleavage of the RNA well away from the catalytic center. By this model, the nascent RNA enters a product binding site beginning 3 or 4 nucleotides from the growing point at the 3' terminus. This RNA binding site extends for up to 16 nucleotides along the protein surface. The stress brought about by this binding appears to vary considerably for different ternary complexes and may play a role in driving the translocation of the RNA polymerase along the DNA.
Surratt
C K
CK
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Milan
S C
SC
Chamberlin
M J
MJ
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli; D006868:Hydrolysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases