Template end-to-end transposition by RNA polymerase II

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• RNA polymerase II (Pol II) performs promoter-initiated transcription.
• Run-off transcription generates products longer than expected on specific templates.
• Elongation factor SII (SII) is known to affect transcription elongation.

Purpose of the Study:

• Investigate the mechanism of extended transcript production during run-off transcription.
• Determine the role of template terminal complexes in generating these extended RNAs.
• Elucidate the function of elongation factor SII in transcript extension and template switching.

Main Methods:

• In vitro run-off transcription assays using various template designs (5'-overhang, blunt-ended).
• Analysis of RNA products generated by RNA polymerase II.
• Assessment of transcript extension and its salt sensitivity.
• Evaluation of elongation factor SII's effect on transcript extension and complex stability.

Main Results:

• Template terminal complexes produce discrete, extended run-off transcripts (15-16 nt longer).
• Transcript extension is a general, salt-sensitive feature of run-off transcription.
• Elongation factor SII facilitates transcript extension on 5'-overhang templates via a dinucleotide truncation intermediate.
• Extended complexes on one 5'-overhang template showed spontaneous, salt-resistant extension.
• On blunt-ended templates, SII addition resulted in a 4-5 nt extension product.

Conclusions:

• Extended run-off transcripts represent intermediates and successful completion of template end-to-end transposition (template switching) by RNA polymerase II.
• Template end sequence and structure influence the success of template switching.
• Elongation factor SII plays a crucial role in facilitating transcript extension and potentially template switching.