Polymerase processivity and termination on Drosophila heat shock genes

Area of Science:

• Molecular Biology
• Gene Regulation
• Biochemistry

Background:

• RNA polymerase II transcription is regulated at multiple steps.
• The drug 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is known to inhibit transcription.
• The Drosophila hsp70 gene exhibits a paused polymerase at its 5'-end in the uninduced state.

Purpose of the Study:

• To investigate the mechanism of DRB-induced transcriptional pausing.
• To compare DRB-sensitive steps with the natural pausing of polymerase at the hsp70 gene.
• To analyze the polymerase termination process at the hsp26 gene.

Main Methods:

• In vivo UV cross-linking.
• KMnO4 hyperreactive site mapping.
• Polymerase-DNA cross-linking.

Main Results:

• DRB treatment allows transcription to proceed beyond the naturally paused polymerase site at hsp70.
• DRB treatment reduces hyper-reactivity in regions associated with open polymerase and early elongation complexes.
• Polymerase termination at the hsp26 gene occurs at multiple downstream sites.

Conclusions:

• The DRB-sensitive transcriptional block is distinct from the natural pausing mechanism at the hsp70 gene's 5'-end.
• DRB likely targets a polymerase modification step affecting early elongation.
• Multiple termination sites exist downstream of the polyadenylation signal for hsp26.