Structure and function of the initially transcribing RNA polymerase II-TFIIB complex

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• General transcription factor IIB (TFIIB) is crucial for RNA polymerase (Pol) II initiation.
• Previous low-resolution structures lacked nucleic acids and parts of TFIIB, limiting understanding of its functions.
• TFIIB's role in DNA recruitment was known, but other functions remained unclear.

Purpose of the Study:

• To determine high-resolution crystal structures of the Pol II-TFIIB complex.
• To elucidate the role of the entire B-reader element in transcription initiation.
• To understand the detailed molecular mechanisms of TFIIB in RNA synthesis and product release.

Main Methods:

• X-ray crystallography of Saccharomyces cerevisiae Pol II-TFIIB complex at 3.4 Å resolution.
• Structural analysis of an initially transcribing complex with DNA and a 6-nucleotide RNA product.
• Integration of structural data with functional assays.

Main Results:

• Revealed the complete B-reader structure and its interactions with protein and nucleic acids.
• Demonstrated TFIIB's role in closing the polymerase cleft, positioning DNA, and facilitating DNA opening.
• Showed TFIIB allosterically stimulates RNA synthesis by rearranging active-site residues and inducing metal ion binding.
• Identified TFIIB's function in preventing DNA-RNA hybrid tilting and directing RNA through its exit tunnel.
• Described TFIIB displacement upon RNA growth to 12-13 nucleotides, triggering elongation complex formation.

Conclusions:

• TFIIB plays multifaceted roles in transcription initiation, from DNA positioning to RNA product release.
• The B-reader is critical for initiator recognition, transcription start site positioning, and RNA processing.
• TFIIB's mechanism explains fundamental differences between RNA and DNA polymerases, such as primer-independent initiation.
• Similar mechanisms involving TFIIB-like factors are likely conserved across eukaryotic and archaeal transcription.