Department of Chemistry, University of California, Berkeley.
Biochemistry 29: 7882-90 (1990)
Abstract
We have used a self-cleaving RNA molecule related to a subsequence of
plant viroids (a "hammerhead") to study the length-dependent folding of
RNA produced during transcription by Escherichia coli RNA polymerase.
Transcript elongation is arrested at defined positions using
chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's
or 3'-O-methylNTP's). When the transcript can form the "hammerhead"
structure it self-cleaves to give a truncated product. The experiment
yields an RNA sequencing ladder which terminates at the length at which
cleavage becomes possible; the sequencing ladder is compared to those
generated by using a noncleaving transcript or by using [alpha-thio]ATP
in place of ATP. We have shown that 15-18 nucleotides (nt) of RNA past
the cleavage point must be synthesized before the transcript can
self-cleave within a ternary complex, whereas RNA freed from the complex
by heating can cleave with only 3 or more nt present beyond the
cleavage point. There are sequence-dependent as well as length-dependent
effects. The results suggest that 12 +/- 1 nt are sequestered within
the ternary complex and are consistent with the presence of a DNA-RNA
hybrid within the transcription bubble, as proposed by others. The
results indicate that the "hammerhead" structure does not disrupt the
hybrid. It appears that the RNA beyond the hybrid is not restrained by
interactions with the enzyme, since the last stem of the self-cleaving
structure forms as soon as the RNA composing it emerges from the DNA-RNA
hybrid. Self-cleaving of the transcript offers a simple structural
probe for studying less well-characterized transcription complexes. The
relevance of the results to models for transcription termination is
discussed.
Mesh Headings
Unique Identifier: 91084449
Chemical Identifiers (Names)