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Figure 5.
Figure 5. Intrinsic RNA Cleavage Activity and Functional
Architecture of Pol I
(A) DNA-RNA hybrid scaffold used in
cleavage assays.
(B) Comparison of RNA cleavage by Pol I
variants with Pol II and the Pol II-TFIIS complex. Pol I mainly
removed four nucleotides from the RNA, consistent with binding
of the terminal hybrid base pair to the nucleotide insertion
site (+1), extrusion of the RNA 3′ overhang into the
polymerase pore and cleavage of the phosphodiester bond between
nucleotides at positions −1 and +1 (Figure 5A). The Pol
II-TFIIS complex removed three or four nucleotides, indicating
that a mixture of complexes was present with the terminal hybrid
base pair occupying either position −1 or +1.
(C) pH
dependence of pol I cleavage activity.
(D) Elongation
activity of the Pol I variant A12.2ΔC.
(E) Hybrid
structure and functional architecture of Pol I. The EM envelope
is shown as a blue line, the Pol I core ribbon model in gray
with Rpb9 (A12.2) highlighted in orange, and the A14/43 crystal
structure in red/blue. The window shows a cut-away view of the
active center containing a modeled DNA-RNA hybrid. Red dashes
indicate the RNA 3′ end extruded into the pore.
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