Probing The Structure And Function Of Transcription Complex Of RNA Polymerase II With TFIIF At Single Molecular Level

Abstract

Previously, we have developed a bio-conjugation method that allows us to specifically label the C-terminus of any subunit of a RNA polymerase. Here, we reconstitute a RNA polymerase II (Pol II) with TFIIF (IIF), in which the C-terminus of a RNA polymerase subunit is labeled with Cy3 and an amino acid of the largest subunit of TFIIF is labeled with Cy5. By measuring FRET at single molecular level, we are able to probe the structure information of Pol II/IIF in the absence of X-ray structure. By using single molecular FRET and tethered particle motion method, we are also carrying out transcription of Pol II in the presence or absence of IIF to identify the role of IIF in RNA elongation at various stages.