Concerted Protein and Nucleic Acid Conformational Changes Observed Prior to Nucleotide Incorporation in a Bacterial...

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Spectroscopy

Background:

• Transcription elongation by RNA polymerases (RNAPs) involves processive nucleotide incorporation and enzyme translocation.
• Understanding the dynamic conformational changes during nucleotide addition is crucial for deciphering RNAP function.
• Previous work utilized Raman spectroscopy and gel electrophoresis to study Thermus thermophilus RNAP elongation complexes (TthEC).

Purpose of the Study:

• To investigate the time-resolved conformational dynamics of the TthEC during single-nucleotide incorporation using Raman spectroscopy.
• To correlate structural changes in protein and nucleic acids with the catalytic event of nucleotide addition.
• To explore the binding and reactivity of GTP within the TthEC crystal.

Main Methods:

• Time-resolved Raman spectroscopy was employed on single crystals of the Thermus thermophilus elongation complex (TthEC).
• Isotopically labeled GTP ([ (13)C,(15)N]GTP) was soaked into TthEC crystals to monitor nucleotide incorporation.
• Analysis focused on spectral changes assigned to protein and nucleic acid conformational events and GTP vibrational modes.

Main Results:

• Large, reversible Raman spectral changes indicated protein and nucleic acid conformational rearrangements during nucleotide incorporation.
• Protein conformational changes, particularly in α-helical regions, peaked around 50 minutes post-GTP addition.
• Catalysis, monitored by GTP triphosphate vibrational mode decline, initiated around 65-70 minutes, after maximal structural changes.

Conclusions:

• The study reveals a distinct temporal sequence where structural dynamics precede the catalytic incorporation of GTP in TthEC.
• Approximately 40% of the incorporated GTP reacted, suggesting a potential second, non-catalytic binding site for GTP.
• The observed slow time regime allows for detailed dissection of structural dynamics during GMP incorporation within the TthEC crystal.