Stability of Escherichia coli Transcription Complexes Near an Intrinsic Terminator

Abstract

Transcription complexes containing Escherichia coli RNA polymerase terminate RNA synthesis in response to intrinsic (rho-independent) terminators encoded in the DNA template. This type of termination involves the formation of a hairpin structure in the nascent RNA, leading to release of the RNA and polymerase from the DNA template. In this paper we show that RNA release occurs only within a zone extending over six template positions of the tR2 terminator of phage λ. The upstream end of this zone is defined by large and abrupt decrease in the stability of the transcription complex, and this decrease is reversed at the downstreame end. Except within this termination zone, RNA chain elogation is completely processive at all template positions, over a wide range of temperatures (20 to 70°). The stability of ternary complexes halted at each template position by the incorporation of 3′-deoxynucleotide substrate analogues was determined by means of an ultrafiltration RNA release assay. This assay shows that the nascent RNA is released from halted ternary complexes within the same zone in which termination occurs during active transcription. The termination efficiency at each position within the tR2 termination zone is a sensitive function of the concentration of the next nucleotide substrate to be incorporated into the nascent RNA. These results suggest that termination positions are defined by the thermodynamic stability of the polymerase complex, while the efficiencies of termination are determined by a kinetic competition between RNA elongation and release at each position. Based on a kinetic competition model , the temparature dependence of the efficiency indicates that the elogation pathway is favored enthalpically, while the termination pathway is favored entropically.