Distributive enzyme binding controlled by local RNA context results in 3' to 5' directional processing of dicistronic tRNA precursors by Escherichia coli ribonuclease P

Abstract

RNA processing by ribonucleases and RNA modifying enzymes often involves sequential reactions of the same enzyme on a single precursor transcript. In Escherichia coli, processing of polycistronic tRNA precursors involves separation into individual pre-tRNAs by one of several ribonucleases followed by 5' end maturation by ribonuclease P. A notable exception are valine and lysine tRNAs encoded by three polycistronic precursors that follow a recently discovered pathway involving initial 3' to 5' directional processing by RNase P. Here, we show that the dicistronic precursor containing tRNAvalV and tRNAvalW undergoes accurate and efficient 3' to 5' directional processing by RNase P in vitro. Kinetic analyses reveal a distributive mechanism involving dissociation of the enzyme between the two cleavage steps. Directional processing is maintained despite swapping or duplicating the two tRNAs consistent with inhibition of processing by 3' trailer sequences. Structure-function studies identify a stem-loop in 5' leader of tRNAvalV that inhibits RNase P cleavage and further enforces directional processing. The results demonstrate that directional processing is an intrinsic property of RNase P and show how RNA sequence and structure context can modulate reaction rates in order to direct precursors along specific pathways.

Figure 1.

3′ to 5′ directional RNase P cleavage is the initial step in processing of three dicistronic tRNA precursors. (A) The eleven polycistronic tRNA precursors are listed that are processed by the major processing pathway in which the initial step is separation into individual monocistronic units by RNase E (indicated by an arrow), 5′ end maturation by RNase P (shown as a lightning bolt), and subsequent 3′ end trimming (not shown). (B) The three polycistronic tRNA precursors encoding the entire complement of valine and lysine tRNAs undergo initial 3′ to 5′ directional processing by RNase P, followed by 3′ end trimming.

Scheme 1.

Consecutive first order reaction.

Figure 2.

In-line structure probing of 5′ ³²P end-labeled dicistronic valVW precursor RNA corresponds to predicted secondary structure. (A) Predicted secondary structures of the valVW substrate. Levels of spontaneous RNA cleavage at backbone are circled and shaded according to the intensity of the cleavage product. Analysis of valVW cleavage products on 12% (B) and 8% (C) polyacrylamide gels. Lanes are marked as follows. UR: unreacted RNA; T1: partial RNase T1 digestion; OH: partial alkaline hydrolysis; VW-ILP: refolded ptRNAs^val VW subjected to in-line probing condition (50 mM Tris–HCl (pH 8.3 at 20°C), 20 mM MgCl₂, and 100 mM KCl) incubated in 21°C for 40 h. Bands corresponding to sites of T1 digestion are numbered as indicated in panel A.

Figure 3.

In vitro processing of of 5′ ³²P end-labeled (A) and uniformly labeled with α-³²P-CTP (C). Reactions either containing or lacking RNase P are marked by plus (+) or minus (–) symbol, respectively. Structures of the substrate and predicted structures of products are shown next to each band based on gel mobility. The tRNAvalV is shown as a black line and the tRNA^valW is shown as a dashed line. Total incubation time for substrate is shown as an oblique triangle (30 minutes). (B). Fitting of the change in fraction of the substrate a, intermediate b, and product c to equations for two sequential first order reactions. As described in Materials and Methods, the data for a are fit to equation 1 is shown as a dashed line, the data for b and c are fit to Equations (2) and (3), respectively.

Figure 4.

Site specificity of RNase P processing of the dicistronic valVW precursor in vitro. Phosphorimager analysis of cleavage products resolved on 8% (A) and 12% gel (B) polyacrylamide gels with 5′ ³²P end-labeled valVW RNA. The lanes are marked as follows. (C) Control reaction with no enzyme, OH: alkaline hydrolysis of dicistronic valVW, T1: RNase T1 ladder. The positions of the guanosines and surrounding sequence are indicated next to the gel.

Figure 5.

Dependence of the observed rate constants and accumulation of reaction intermediate as a function of enzyme concentration. (A) Two possible mechanisms for RNase P processing of dicistronic ptRNAval VW. In the first model the processing of the two site is processive (Scheme 1, processive) without dissociation of the enzyme between the two cleavage events, and the second is a distributive model (Scheme 2, non-processive) ) in which processing at the two sites occurs independently. (B) Dependence of k₁ and k₂ for 3′ to 5′ directional processing of valVW on RNase P concentration under single turnover reaction conditions (Materials and Methods). (C) Comparison of the accumulation intermediate b in reactions containing high (60 nM) and low (3 nM) enzyme concentration showing accumulation to the same extent independent of enzyme concentration.

Figure 6.

Determination of the reversibility of binding at the 5′ tRNA valV processing site. (A) Time courses of processing of 5′ ³²P-labeled dicistronic valVW precursor by RNase P under single turnover conditions without (left) or with (right) addition of excess unlabeled valVW substrate after ∼30% of radiolabeled substrate had been cleaved at the 3′-proximal site. (B and C) The kinetics of substrate a, intermediate b and 5′ leader fragment c are fit to equations for two sequential first order reactions for both data sets to illustrate the change in reaction kinetics post-chase.

Figure 7.

Kinetics analysis RNase P processing of 5′ ³²P-labeled dicistronic valWV (A and B), valVV (C and D) and valWW (E and F) substrate RNAs. Single turnover reactions were performed as described for the native valVW substrate. The position of cleavage resulting from processing at the correct sites is supported by T1 mapping (see Supplementary Figures S3–S5). The structure of substrates and products are shown next to each band. The tRNAvalV is shown as a black line and the ptRNAvalW is shown as a dashed line. Incubation time for each substrate is shown as an oblique triangle (30 min). Panels B, D and F show quantitative analysis by fitting the data for each reaction to equations for sequential first order reactions as described in Material and Methods.

Figure 8.

In-line structure probing of 5′ end labeled dicistronic valWV (A, B), valVV (C, D) and valWW (E, F) substrate RNAs. Panels A, C and E show the levels of spontaneous RNA cleavage at patterns observed with 5′ ³²P end-labeled RNAs resolved on 12% and 8% polyacrylamide gels. Lanes are indicated above the gel as follows. NR: unreacted RNA; T1: partial RNase T1 digestion; OH: partial alkaline hydrolysis; WV/VV/WW-ILP: in-line probing reaction. Bands corresponding to certain T1 digestion and alkaline hydrolysis are circled on the predicted secondary structures of valWV, valVV, and valWW in panels B, C and D, respectively.

Figure 9.

Single turnover reaction analysis of precursor tRNA rate constants. (A) Comparison of rate constants for ptRNA^valV and ptRNA^valW in monocistronic versus dicistronic contexts. The substrate structures are shown on the left. The ptRNA^valV substrate is shown as a dashed line and the ptRNA^valW is shown as a solid line. (B) Analysis of the effects of leader sequence structure on RNase P processing. The predicted secondary structure of the leader sequences of the individual ptRNA^valV mutants are shown on the left.