ausblenden:
Schlagwörter:
X-ray crystallography, Enzyme mechanisms, Transcription
Zusammenfassung:
The general transcription factor (TF) IIB is required for RNA polymerase
(Pol) II initiation and extends with its B-reader element into
the Pol II active centre cleft. Low-resolution structures of the Pol II–
TFIIB complex1,2 indicated how TFIIB functions in DNA recruitment,
but they lacked nucleic acids and half of the B-reader, leaving
other TFIIB functions3,4 enigmatic. Here we report crystal structures
of the Pol II–TFIIB complex from the yeast Saccharomyces
cerevisiae at 3.4A˚ resolution and of an initially transcribing complex
that additionally contains theDNAtemplate and a 6-nucleotide
RNAproduct.The structures reveal the entire B-reader and protein–
nucleic acid interactions, and together with functional data lead
to a more complete understanding of transcription initiation.
TFIIB partially closes the polymerase cleft to position DNA and
assist in its opening. The B-reader does not reach the active site but
binds the DNA template strand upstream to assist in the recognition
of the initiator sequence and in positioning the transcription
start site. TFIIB rearranges active-site residues, induces binding of
the catalytic metal ion B, and stimulates initial RNA synthesis
allosterically. TFIIB then prevents the emergingDNA–RNAhybrid
duplex from tilting, which would impair RNA synthesis. When the
RNA grows beyond 6 nucleotides, it is separated from DNA and
is directed to its exit tunnel by the B-reader loop. Once the RNA
grows to 12–13 nucleotides, it clashes with TFIIB, triggering TFIIB
displacement and elongation complex formation. Similar mechanisms
may underlie all cellular transcription because all eukaryotic
and archaeal RNA polymerases use TFIIB-like factors5, and the
bacterial initiation factor sigma has TFIIB-like topology1,2 and contains
the loop region 3.2 that resembles the B-reader loop in location,
charge and function6–8. TFIIB and its counterparts may thus
account for the two fundamental properties that distinguish RNA
from DNA polymerases: primer-independent chain initiation and
product separation from the template.
