RNA polymerase II is capable of pausing and prematurely terminating transcription at a precise location in vivo and in vitro

Abstract

By using the minute virus of mice, we have shown that in vivo and in vitro RNA polymerase II pauses or prematurely terminates transcription at a specific location 142-147 nucleotides downstream from the P4 promoter. The attenuated RNA was found and mapped in vivo in A9 cell late after infection in both the nuclear and cytoplasmic fractions, and the terminal nucleotide was shown to have a 3' OH group. The 3' end of the attenuated RNA is capable of forming a hairpin structure that is followed by a stretch of uridines. To distinguish whether the attenuated RNA is formed as a result of processing, pausing, or termination and to dissect structural elements, factors, or mechanisms that are involved in its formation, we used in vitro systems: isolated nuclei and cell-free extracts from HeLa cells. The results of the in vitro studies show that the attenuated RNA is a result of pausing or termination and not processing. Additionally, a salt-soluble factor and RNA secondary structure were implicated in the process of termination.