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. 2014 Jun 10;111(23):E2368-75.
doi: 10.1073/pnas.1319740111. Epub 2014 May 22.

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Christal L Vitiello  1 , Maria L Kireeva  2 , Lucyna Lubkowska  2 , Mikhail Kashlev  3 , Max Gottesman  4
Affiliations

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Christal L Vitiello et al. Proc Natl Acad Sci U S A. .

Abstract

The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β' subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5' edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.

Keywords: Bacteriophage HK022; Bacteriophage Lambda; Exclusion; Transcription Elongation; Transcription Termination.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Assembly of Nun-sensitive TECs. (A) Cartoon representation of TEC assembly. (B) TEC G9 was assembled by addition of a 32P 5′-labeled RNA9 hybridized to a TDS65, followed by addition of NDS65. TEC A11 was obtained by addition of ATP and UTP (5 μM each) to the G9 assembly reaction. G9 and A11 were purified by membrane filtration. TECs U10 and C12 were obtained from G9 and A11 by 5 min of incubation with 10 μM UTP or CTP, respectively. TECs were then preincubated for 10 min at 25 °C with 5 μM Nun, or a comparable volume of Nun storage buffer before transcription was initiated by addition of 1 mM NTPs and stopped with an equal volume of 2× stop buffer. The 0-, 10-, and 60-s time points are shown.
Fig. 2.
Fig. 2.
Nun effects on RNAP toe prints. (A) Rear-end toe printing of TECs A11 and C12 in the presence and absence of Nun. Upstream DNA was unidirectionally digested with ExoIII to the rear-end boundary of each TEC (the TDS was 5′-labeled). Reactions were stopped after 2 min of incubation. (Upper) Toe prints (including the length of the DNA fragments) are indicated by arrows on the immediate right side of the blot. (Right) Cartoon indicates how ExoIII digestion defines the pre- and posttranslocated states of TEC as determined by the rear-end toe print. (Lower) RNA synthesized by each TEC is shown. (B) Front-end toe printing of TECs A11 and C12. Downstream DNA was unidirectionally digested with ExoIII. Posttranslocated boundaries of A11, C12, and G9 (a translocation marker) are indicated by diagonal arrows next to the corresponding gel lanes. (Upper) Lengths of the ExoIII digestion products are indicated on the immediate right side of the blot. (Lower) RNA products are again shown. (Right) Cartoon illustrates front-end ExoIII digestion.
Fig. 3.
Fig. 3.
RNAP β′ mutations near the upstream edge of the RNA–DNA hybrid inhibit Nun. (A, Left) Structure of 9-bp RNA–DNA hybrid in a TthRNAP TEC is shown (PDB ID code 2O5J). All three RNAP mutants that inhibit arrest by Nun in vivo locate in the β′ subunit of EcRNAP (β′ D264G, β′ D329G, and β′ R322H) between the lid and rudder domains in the zipper region. The mutations are clustered within 3–6 Å of the 5′ end of the transcription bubble in the vicinity of base pair −9 of the 9-bp RNA–DNA hybrid. Only the DNA (gray, space-filled model) and the RNA (colored elements, sticks) strands of the 9-bp RNA–DNA hybrid and the NTP in the active center (i + 1 site) are shown. Numbers in brackets indicate the corresponding amino acid residues in the β′ subunit of TtRNAP. (Inset) Zoomed area surrounding the −8/−9 bp of the hybrid. The −8 RNA and DNA paired bases (−8R and −8D) are shown in yellow and brown colors, respectively. The R322 (TtRNAP R598) residue is located 3 Å from the α-phosphate of the −8 residue of the nascent RNA. (A, Right) Model of the structure of the “complete” TEC by yeast RNAPII containing the intact transcription bubble (44). The colors are as in the left panel. (Inset) The −8 and −9 positions of the RNA–DNA hybrid are highlighted; the position of Rpb1-R320 (corresponds to EcRNAP β′ R322) is shown. (B) TECs A11 were assembled with WT and β′ R322H mutant RNAPs immobilized on Ni2+-NTA agarose for the TEC purification with RNA11, TDS65, and NDS65. C12 TECs were obtained by 90 s of incubation with 5 μM CTP. A nonfunctional mutant of Nun (K106/107A) was tested with WT TEC (lane 9). The +11A RNA is indicated by an arrow. (C) Upstream DNA was unidirectionally digested with ExoIII to the rear-end boundary of A11 or C12 TECs. Before ExoIII treatment, each TEC was incubated for 5 min with Nun or a comparable volume of Nun storage buffer. The Nun-specific toe print is indicated on the immediate right side of the blot by an arrow. (Right) Cartoon indicates the location of the Nun-specific toe print.
Fig. 4.
Fig. 4.
Presteady-state analyses of Nun effect on NMP addition and pyrophosphorolysis. (A) CMP addition time course by TEC A11. TEC A11 was obtained as described in Fig. 1B. Transcription was assayed by mixing the TEC with ATP and CTP (1 mM final concentration) using an RQF-3 rapid quench flow instrument (Kintek). The fraction of the 12-nt or longer products was plotted vs. time, and the data were fitted with a double-exponential function. The apparent rates and fractions of the TECs incorporating the CMP with these apparent rates are shown in the plot. (B) Pyrophosphorolysis of TEC A11. The TEC was incubated with 2.5 mM potassium pyrophosphate for the indicated times. The reaction was stopped manually by addition of gel-loading buffer. The fraction of the 11-nt RNA product was plotted vs. time, and the data were fitted with a single exponential function. (C) Proposed mechanism of Nun interaction with TEC A11. (D) AMP addition time course by TEC C12. The experiment was performed and analyzed as described in A, except that TEC C12 was obtained from TEC A11 before Nun addition. (E) Pyrophosphorolysis of TEC C12. The experiment was performed and analyzed as in B, but the reaction was carried out with an RQF-3 rapid quench flow instrument. (F) Nun effect on susceptibility of TEC C12 to pyrophosphate. The fraction of TEC C12 remaining intact after 5 s of incubation of TEC C12 with various pyrophosphate concentrations is plotted. The data were fitted to a Michaelis–Menten equation to determine the concentration of pyrophosphate promoting pyrophosphorolysis of 50% of the elongation complex after 5 s of incubation with pyrophosphate. Δ t (s) (time in seconds), threshold cycle.
Fig. 5.
Fig. 5.
Hybrid length determines the ability of Nun to inhibit transcription. TECs were assembled as described in Fig. 1, but with RNA8A, RNA9A, and RNA11A (Table S1). All of the RNA primers shared the 3′ sequence, ending at +11A. RNA11A was fully complementary to TDS65, and RNA11AM carried a 3-nt 5′-end mismatch. TEC 11AR11 was assembled with RNA11AM, TDS65M, and NDS65M, restoring the full complementarity of the RNA to the template. The assembled TECs were preincubated with Nun or Nun storage buffer before (A) or after (B) addition of 5 μM CTP for 5 min. NTPs (1 mM) were added in even-numbered lanes for 60 s.
Fig. 6.
Fig. 6.
Abasic sites in the TDS that disrupt the RNA–DNA hybrid block Nun-mediated transcription arrest and Nun binding to TEC. (A) TEC 9A11 was assembled with RNA9A; original TDS65; or TDS65 containing abasic substitutions −8, −9, and −10 relative to the location of the RNA 3′ end (TDS65 abasic series; Table S1) and NDS65. AB, abasic. (B) Rear-end boundary of TEC 9A11 on the original, −8 abasic, −9 abasic, and −10 abasic templates was determined by ExoIII toe printing. A short arrow indicates the position of the Nun-specific toe print. Bars and cartoon indicate the positions of pre- and post-translocated TEC 9A11. (C) TEC 9A11 was assembled with RNA9A; TDS65; original NDS65; or NDS65 containing abasic substitutions −8, −9, and −10 relative to the location of the RNA 3′ end (NDS65 abasic series; Table S1). Transcription assays in A and C were performed as described in Fig. 3B.
Fig. 7.
Fig. 7.
Molecular mechanism of Nun action. Details are provided in the main text.
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