Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide...

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Bacteriophage T7 RNA polymerase (RNAP) undergoes significant conformational changes during transcription initiation to elongation.
• Understanding these dynamic transitions is crucial for elucidating transcription regulation mechanisms.

Purpose of the Study:

• To investigate the functional significance of RNAP conformational changes during transcription.
• To analyze the effects of restricted conformational mobility on transcription initiation, elongation, and termination.

Main Methods:

• Construction of mutant T7 RNAPs with disulfide bonds to limit mobility of key elements.
• Analysis of transcription initiation, elongation, pausing, and termination using these crosslinked RNAPs.
• Characterization of crosslink effects on different terminator classes (I and II).

Main Results:

• A crosslink specific to the initiation complex blocked transcription early, preventing isomerization to the elongation complex (EC).
• A crosslink specific to the EC minimally affected elongation and class I terminator (T) termination.
• Crosslinked ECs showed normal pausing at class II pause sites but were deficient in termination at class II terminators (e.g., PTH).

Conclusions:

• RNAP conformational flexibility is essential for efficient transcription initiation and class II terminator function.
• Specific crosslinks highlight the distinct mechanisms of class I and class II termination.
• The results suggest that polymerase movement near the RNA-DNA hybrid is critical for releasing RNA at certain terminators.