RNA synthesis in isolated nuclei: the use of mercurated nucleotides

Abstract

We have used uridine 5' triphosphate-5-mercury (Hg-UTP) in place of UTP to study RNA synthesis in a previously described isolated nuclei system (1). Employing isopycnic density gradient centrifugation to separate RNAs based upon their relative content of Hg-U, several conclusions can be drawn. In vitro RNA synthesis consists of end addition onto pre-initiated HnRNA molecules as well as apparent initiation of new HnRNA molecules de novo. Synthesis in our system continues linearly for greater than two hours. The chain elongation rate has been measured to be about 500 nucleotides per minute. The methods used to make these measurements are generally applicable to other in vitro systems.