Nature, 2 February 2006: Vol 439: 4337

The mechanism of DNA replication primer

synthesis by RNA polymerase
1,3 1 1 1,2,3
Nikolay Zenkin , Tatyana Naryshkina , Konstantin Kuznedelov & Konstantin Severinov

1
Waksman Institute,
2
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey
3
Institute of Molecular Genetics, RussianAcademy of Sciences, Moscow
Structure of the M13 origin of replication.
pRNA is shown as an arrow
 C, pRNA was synthesized using RNAP immobilized on Ni-
NTA agarose. The bead suspension (t) was separated into
a pellet (p) and the supernatant (s) and the contents were
resolved by urea–PAGE.
d, pRNA containing a crosslinkable group at its 5’ end
was synthesized, the crosslink was induced and reaction
products were separated by SDS–PAGE and revealed by
protein staining and autoradiography.

Figure 1 | Properties of the priming complex. b, A gel-

filtration elution profile of radioactive pRNA synthesis A ternary complex other than
reactions not treated (black squares) and treated (empty
squares) with RNase H. Fractions were analysed by SDS–
normal transcription event results
PAGE for protein content and by urea-PAGE for pRNA. In in the formation of pRNA
the lower panel, fractions were treated with RNase H. A black
arrow in the plot indicates the elution volume of pRNA from
deproteinized pRNA synthesis reaction. AP, abortive
products; CP, cleavage products.
 Comparison of the properties of the priming complex (lanes 11–15), the active elongation complex
(TEC20, lanes 1–5), and the backtracked elongation complex (TEC27, lanes 6–10). RO, run-off;
T, terminator. PPi, pyrophosphate.

pRNA ternary complex is a DNA-RNA hybrid and has exposed 3’ –OH end
 Figure 2 | Overextended RNA–DNA hybrid causes formation of the priming complex. a, pRNA synthesis was
performed on the wild-type (wt, lanes 1and 3) origin fragment or a mutant fragment containing dAMPs at positions
þ2, þ3 and þ4 of the template strand instead of dCMP (Mut, lane 2) in the presence of GTP (lanes 1 and 2) or ITP
(lane 3). RO, run-off. b, The RNA–DNA crosslink from the 5’ end of the RNA in stalled complexes, obtained as
described in Methods, with RNAs of the indicated lengths, containing either GMP (lanes 1–8) or IMP (lanes 9–16).

Strength of base pairing between 5’ of RNA and template DNA controls the
pRNA formation
 pRNA synthesis by wild-type RNAP (lane 1) and by mutant RNAPs lacking the lid (Lid, lane 2), the zipper
(Zipper, lane 3) or the rudder (Rudder, lane 4).

Collision of the 5’ end of the nascent RNA with the ’ lid leads to the formation of
overextended hybrid
 Trajectory of pRNA

Figure 3 | Structure of the priming complex.

a, Mapping of the crosslink from the 5’ end of pRNA. The b subunit radioactively labelled bycrosslinking to the 5’ end of pRNA in the
priming complex (lanes 1–3) was treated with CNBr under single-hit conditions for different durations and the cleavage products
were separated by SDS–PAGE. As a marker, the b subunit affinity labelled on Lys 1065 (ref. 15) and subjected to identical CNBr
treatment was used (lanes 4–6). Solid lines connect corresponding cleavage products from the 10-min lanes.
b, T7 gp2 destroys the priming complex. pRNA synthesis reactions were performed on the solid phase. After incubation with or
without gp2, the reaction mixtures (t, lane 1) were divided into supernatant (s, lanes 3, 5 and 6) and pellet (p, lanes 2 and 4); in lane
6 the supernatant was treated with RNase H. CP, cleavage products.
 a RNA exit channel
b
’ lid
downstream DNA RN 1
binding channel Mg2+ A

5’ 3’
downstream DNA upstream DNA
stable elongation
complex
NTPs
NTPs

NTPs NTPs

5’ 3

3’ Priming complex
5’