Analyzing the Dynamics of Single TBP-DNA-NC2 Complexes Using Hidden Markov Models

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Single-pair Förster resonance energy transfer (spFRET) is crucial for studying biological conformational dynamics.
• Existing hidden Markov model (HMM) approaches require refinement to accurately capture spFRET data complexities.

Purpose of the Study:

• To develop an enhanced HMM analysis for spFRET data that incorporates photon-shot noise.
• To investigate the dynamics of TATA-box-binding protein (TBP) complexed with negative cofactor 2 (NC2) on promoter DNA.

Main Methods:

• Extended the hidden Markov model (HMM) by incorporating photon-shot noise into the analysis.
• Applied the enhanced HMM-spFRET approach to study TBP-DNA-NC2 complex dynamics on different promoter sequences and DNA constructs.
• Utilized clustering of FRET states to compare dynamics across different experimental conditions.

Main Results:

• Identified four distinct FRET states for TBP-DNA-NC2 on the adenovirus major late promoter, corresponding to conformational changes and complex translocation.
• Observed more complex dynamics for the H2B promoter, with a significantly more stable bent conformation compared to the adenovirus major late promoter.
• Demonstrated that the transition dynamics from stretched to bent DNA conformations are similar for both promoters.

Conclusions:

• The enhanced HMM analysis provides a more accurate method for extracting dynamic information from spFRET data, accounting for experimental noise.
• The study reveals distinct dynamic behaviors and conformational stabilities of TBP-DNA-NC2 complexes on different promoter DNA sequences.
• This refined analytical approach advances the capability to compare molecular dynamics across various biological contexts.