Termination of transcription by RNA polymerase III from wheat germ

A novel system has been established to study transcription by RNA polymerase III. Purified wheat germ RNA polymerase III will initiate transcription on SV40 DNA templates amplified by polymerase chain reaction. Transcription initiates primarily at a thymidine 3 residues from a PvuII terminus at nucleotide 272 and terminates at a downstream A8:T8 sequence at nucleotides 21-28. Termination is enhanced if the RNA polymerase is caused to pause downstream of the A8:T8 sequence, but termination is unaffected by upstream sequences. Transcription of heteroduplex DNA templates with mismatches in the A8:T8 sequence indicates that termination is dependent upon the integrity of the A8 sequence in the template strand and is only slightly affected by changes in the T8 sequence of the non-template strand. However, templates containing 2'-deoxyuridine or 5'-bromo-2'-deoxyuridine in place of thymidine in the non-template strand reduce termination efficiency, as does incorporation of 5'-bromouracil into RNA. There is no obvious correlation between DNA bending and termination efficiency.