Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72 - PubMed (original) (raw)

O G Rössler et al. Nucleic Acids Res. 2001.

Abstract

RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion. Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps. The two proteins resemble each other also with regard to other biochemical properties. We have found that both proteins exhibit an RNA annealing in addition to their helicase activity. By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities. RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis. The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.

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Figures

Figure 1

Figure 1

RNA helicase activity analysis of recombinant p68 and p72. (A) SDS–PAGE (10%) analysis of crude extracts (20 µg each, lanes 1 and 3) and purified p68 and p72 (0.5 µg each, lanes 2 and 4, respectively) isolated from Sf9 cells infected with the respective recombinant baculoviruses. The gel was stained with Coomassie blue. Quantitative determination with a Bioanalyser (Agilent Technologies) using the Protein 200 LabChip Kit revealed 95 and 92% homogeneity of purified p68 and p72, respectively. (B) RNA helicase activity of p72 and p68. Helicase reactions were run with a 17 bp substrate RNA (12.5 nM) plus 0.7 nM (lane 3) or 1.25 nM (lanes 4 and 5) p72, or 1.25 nM (lane 6) or 2.5 nM (lanes 7 and 8) p68 for 15 min at 37°C. Control reactions performed without protein (control, lane 1) or without ATP (lanes 5 and 8) are also shown. One reaction mixture was heat denatured at 95°C before gel electrophoresis (lane 2). (C) Restriction of the unwinding on small dsRNA regions suggests low processivity of the helicases. Helicase reactions were run without protein (lanes 1 and 7) or with p72 (1.25 nM; lanes 3, 5 and 9) or p68 (2.5 nM; lanes 4, 6 and 10) and a 25 (lanes 1–6) or 41 bp (lanes 7–10) substrate (12.5 nM each) as described in (B). Reactions in lanes 5 and 6 additionally contained 6.25 µM of poly(C) as a trap RNA that could be replaced with the same effect by in vitro transcribed, unspecific RNA. RNA helicase activity analysis in (B) and (C) was monitored by gel-shift electrophoresis and autoradiography. A diagram of the substrates used is given on top of each part with an asterisk marking the labelled strand. Note that the upper strand of the 41 bp RNA is identical to that of the 17 bp RNA.

Figure 2

Figure 2

p68- and p72-catalysed RNA annealing. A mixture of two RNA chains (127 and 130 bp long, the latter 32P-labelled) with a 51 bp complementary region was used as substrate. RNA annealing activity was monitored by gel-shift electrophoresis and autoradiography. (A) Dependence of RNA annealing on protein concentrations. Annealing reactions were incubated at increasing concentrations of the indicated proteins (lanes 2 and 6, no protein; lanes 3 and 7, 0.4 nM; lane 4 and 8, 2 nM; lanes 5, 9 and 10, 4 nM; of p72 or p68, respectively) at 37°C for 30 min. The reaction shown in lane 10 additionally contained 1 µg of PAb 204. (B) Dependence of RNA annealing on time. The autoradiogramm illustrates the annealing of RNA complementary strands by p68 (3 nM). Reactions were run for 0 (lane 3), 0.2 (lane 4), 0.5 (lane 5), 1 (lane 6), 2 (lane 7), 5 (lane 8), 10 (lane 9) and 20 min (lane 10). A control reaction run without protein for 5 h is also shown (lane 2). Products of the annealing reactions were run in parallel with the hybrid RNA obtained by hybridisation of the two chains in 80% formamide at 50°C [51 bp RNA, lane 1 in (A) and (B)]. (C) Kinetic plots of p68-dependent and -independent RNA annealing.

Figure 3

Figure 3

Biochemical activities of p68 deletion mutants. (A) RNA-binding activity. Isolated deletion mutants p681–189, p681–386 and p68387–614 were analysed for binding by the retention of 32P-RNA on nitrocellulose filters. Reaction mixtures contained 1 pmol RNA plus the indicated amounts of protein and were incubated for 30 min on ice before filtration. The insert shows SDS–PAGE (12%) analysis of the isolated proteins (0.5 µg each) visualised by Coomassie blue staining. (B) RNA-annealing activity. Annealing activity assays with the indicated amounts of respective proteins were performed exactly as described in (A).

Figure 4

Figure 4

ATP-independent RNA strand exchange. (A) Design of substrate RNAs (with an asterisk marking the labelled strand) and postulated course of the strand exchange reaction. Strand exchange was performed as an intermolecular process between a partially dsRNA and ssRNA, but may also proceed intramoleculary in a similar way. Thick lines indicate homologous regions. Note that the branch migration structure, implicated as a reaction intermediate, is unstable due to the small length (17 bp) of the homologous ds part. (B) ATP-independent strand exchange catalysed by p68 and p72. Reaction mixtures contained the partially ds 17 bp RNA (12.5 nM) plus the respective homologous ssRNA (37.5 nM) and were incubated at 37°C for 30 min at increasing concentrations of p68 or p72 (lanes 4 and 7, no protein; lanes 5 and 8, 2 nM; lanes 6 and 9, 4 nM). For comparison, the 32P-labelled RNA strand (lane 2) and the 17 (lane 1) and 51 bp (lane 3) RNAs prepared by hybridisation of the corresponding RNA strands in 80% formamide at 50°C are shown.

Figure 5

Figure 5

ATP-dependent RNA branch migration catalysed by p72. (A) Formation of a stable RNA branch migration structure in the absence of ATP. To the left (lanes 1–4), RNA helicase reactions (see Fig. 1C) with the partially ds 76 bp RNA as a substrate [with one strand, strand II of the design in (C), being 32P-labelled] are shown. Probes were incubated either without protein (lane 2) or with 2 nM (lane 3) or 4 nM (lane 4) p72 at 37°C for 30 min. To the right (lanes 5–8), the same labelled 76 bp RNA (12.5 nM) plus the non-labelled homologous ssRNA [strand III of the design in (C); 37.5 nM] were used as a substrate in strand exchange reactions without ATP (lanes 5–7) or with PCP (4 mM, lane 8), incubated without protein (lane 5) or at 1 (lane 6) or 2 nM (lanes 7 and 8) p72 for 30 min at 37°C. For comparison, the 123 bp RNA prepared by hybridisation of the respective RNA strands [strands II and III of the design in (C)] in 80% formamide at 50°C are shown (lane 9). (B) Time course of p72-catalysed and protein-independent formation of the stable RNA branch migration structure. Strand exchange reactions without ATP were performed in the presence (2 nM; for 0.5, 1, 2, 5, 10, 15 and 20 min) or absence of p72 (0, 10 and 20 min) exactly as described in (A). Autoradiographic signals of labelled RNA in branch migration structures were quantified with a densitometer (Molecular Dynamics). (C) ATP-dependent branch migration catalysed by p72. As a substrate, we used the same RNAs as in lanes 5–8 of (A) with a different strand being 32P-labelled in each column (strand I, lanes 1–4; strand II, lanes 5–8; strand III, lanes 9–11; see also the design of the reaction at the bottom). Strand exchange reactions were performed with p72 (2 nM) in the absence of ATP for 15 min exactly as described above [lanes 5–8 in (A)] and samples were either analysed directly thereafter (lanes 3, 7 and 10) or after an additional incubation in the presence of ATP (4 mM) for 15 min at 37°C (lanes 4, 8 and 11). Some probes were heat denatured instead of incubated (lanes 1 and 5) or were incubated without protein (lanes 2, 6 and 9). A design of the RNA strand exchange reaction is shown at the bottom with the individual strands marked by roman numbers, and arrows with asterisks indicating the labelled strand in respective reactions. The formation of joint molecules and RNA strand exchange activity was monitored by gel-shift electrophoresis in (A), (B) and (C).

Figure 6

Figure 6

p68-catalysed branch migration. (A) Strand exchange reaction with an RNA substrate containing a 76 bp homologous region. Reactions were performed with the same labelled substrate used in lanes 5–8 of Figure 5A, but with ATP (4 mM) being present during the whole 30 min incubation time either without protein (lane 2) or at 1.5 nM p68 (lane 3), 3 nM p68 (lane 4), 1 nM p72 (lane 5) or 2 nM p72 (lane 6). One probe without protein was denatured at 95°C (lane 1) and the 123 bp RNA formed under hybridisation conditions was run in parallel (lane 7). (B) Strand exchange reaction with an RNA substrate containing a 44 bp homologous region. As a substrate a 44 bp partially dsRNA plus a respective homologous single strand was used, the structure of which was otherwise similar to that of the 76 bp substrate (Materials and Methods). RNA strand exchange activity analysis was performed as described above at 1.5 (lane 4) or 3 nM (lanes 5 and 6) p68 with or without ATP as indicated.

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