Factor-dependent transcription termination by vaccinia RNA polymerase. Kinetic coupling and requirement for ATP hydrolysis

Abstract

Transcription termination during synthesis of vaccinia early mRNAs occurs downstream of a UUUUUNU signal in the nascent transcript and requires a virus-encoded termination factor (VTF), which is identical with the vaccinia mRNA capping enzyme. Using purified transcription complexes halted at defined sites on linear DNA templates, we have examined the order and timing of events during a single round of elongation and termination. We find that although cap synthesis occurs by the time the nascent RNA is 31 nucleotides long, capping enzyme is not stably associated with the elongation complex at this stage. Stable interaction, defined by the formation of a termination-competent complex, requires a longer nascent RNA, e.g. 51 nucleotides, but does not depend on prior transcription of the termination signal. The acquisition of termination competence correlates temporally with the physical association of capping enzyme/VTF with the elongation complex, as revealed by UV cross-linking of the capping enzyme large subunit to the nascent RNA chain. Subsequent induction of termination and transcript release by capping enzyme requires energy, specifically the hydrolysis of ATP. The choice of termination site is flexible and is determined by a kinetic balance between the rate of polymerase elongation and the rate of signaling. Signaling rate is related directly to the concentration of hydrolyzable ATP. An apparent lower limit of 33 nucleotides between the 5' boundary of the termination signal and the most proximal termination site implies that the UUUUUNU signal must be extruded from the RNA polymerase before it can be acted upon by VTF. Similarities between VTF-dependent termination and rho-dependent termination underscore an evolutionarily conserved mechanism for RNA signal transduction to the elongating RNA polymerase.