Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis

Abstract

The active center of RNA polymerase can hydrolyze phosphodiester bonds in nascent RNA, a reaction thought to be important for proofreading of transcription. The reaction proceeds via a general two Mg(2+) mechanism and is assisted by the 3' end nucleotide of the transcript. Here, by using Thermus aquaticus RNA polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (TL). We show that the invariant histidine (beta' His1242) of the TL is essential for hydrolysis/proofreading and participates in the reaction in two distinct ways: by positioning the 3' end nucleotide of the transcript that assists catalysis and/or by directly participating in the reaction as a general base. We also show that participation of the beta' His1242 of the TL in phosphodiester bond hydrolysis does not depend on the extent of elongation complex backtracking. We obtained similar results with Escherichia coli RNA polymerase, indicating that the function of the TL in phosphodiester bond hydrolysis is conserved among bacteria.

Fig. 1.

H1242 of the TL is required for hydrolysis of phosphodiester bonds. (A) Structural alignment of the yeast RNAP II elongation complex in a pretranslocated state [Protein Data Bank (PDB) ID code 1I6H (29)] and the T. thermophilus elongation complex with folded TL [PDB ID code 2O5J (7)] suggests that Q1242 (Green), R1239 (Blue), and H1242 (Yellow) of the folded TL (Light Brown) may come in close proximity to the scissile phosphodiester bond (Cyan) of RNA in the active center. RNA is shown in red and template DNA in gray. Mg²⁺ ions of the active center are shown as spheres. (B and C) Kinetics of phosphodiester bond hydrolysis in MEC(U) and MEC(A) (Fig. S1), respectively, for WT, ΔTL, Q1242A, R1239A, and H1242A RNAPs. Schematics above the gels show the reactions; the asterisk indicates that RNA is labeled at the 5′ end. Representative gels for WT and ΔTL are shown in the middle of each panel (gels for other mutants are shown in Fig. S2). Fractions of cleaved RNA15 against time were fitted to a single exponential equation (Solid Lines) and were normalized to the predicted amplitude. Reaction rates (s^-1) are shown in the graphs legends.

Fig. 2.

H1242 of the TL directly participates in phosphodiester bond cleavage. (A and B) Mg²⁺ dependences of the hydrolysis of the second phosphodiester bond in MEC(U) and MEC(A), respectively, by WT and H1242A RNAPs. Schematics above the plots show the reactions; the asterisk indicates that RNA is labeled at the 5′ end. The fits of data to the Michaelis–Menten equation are shown as solid lines. The k_cat (reaction rate in saturating Mg²⁺) of the reactions were taken as 1 for clarity. K_M[Mg²⁺] values are shown below the plots. (C) pH profiles of second phosphodiester bond hydrolysis in MEC(U) (Red) and MEC(A) (Blue) complexes (colors correspond to reactions shown in A and B) by WT (Solid Squares and Circles, respectively) and H1242A (Empty Squares and Circles, respectively) RNAPs. (D) The first and the second phosphodiester bond hydrolysis in the elongation complex with the correctly paired 3′ end AMP of the RNA (schematically shown above the gel; EC1 in Fig. S1) by WT, H1242A, and ΔTL RNAPs.

Fig. 3.

Impact of TL catalyzed hydrolysis on the proofreading of transcription. (A) The scheme above the gels shows the experimental setup. RNA in the elongation complex (EC2 in Fig. S1) is labeled at the 3′ end by incorporation of [α³²P]UTP (asterisk in the scheme). The 3′ end labeling of RNA enables the monitoring of misincorporation, readthrough, and proofreading simultaneously. Misincorporation of 1 mM ATP, readthrough in the presence of 100 μM CTP, and proofreading (cleavage of pUpA) by WT and H1242A RNAPs are shown. The cleavage products, migrating slower than a dinucleotide, originate from hydrolytic cleavage in readthrough complexes (with RNA longer than 15 nucleotides) after backtracking (see Fig. 4 and Fig. S3) (30). Products are designated in colors the same as those used in the scheme of the reaction shown above the gels. A black vertical line separates lanes originating from the same gel that were brought together. (B) Graphic representation of the data from A. The color code for the plots is the same as in A.

Fig. 4.

Histidine of the TL is required for RNA hydrolysis irrespective of the extent of elongation complex backtracking, and this function of the TL is conserved among bacteria. (A) Schematics at the top of the panel shows formation of elongation complexes MEC(U)ⁿ backtracked by 1 bp [formed from elongation complex EC6 (Fig. S1)] and MEC(UU)ⁿ backtracked by 2 bp [formed from elongation complex EC5 (Fig. S1)]. Below: Kinetics of hydrolysis of the second and the third phosphodiester bonds in MEC(U)ⁿ and MEC(UU)ⁿ, respectively, formed via 1 mM UTP misincorporation by WT and H1242A RNAPs. A black vertical line separates lanes originating from the same gel that were brought together. (B) Hydrolysis of the second phosphodiester bond in assembled MEC(U) by E. coli EcWT and EcH936A RNAPs. (C) Kinetics of hydrolysis of the second and the third phosphodiester bonds in MEC(U)ⁿ and MEC(UU)ⁿ, respectively, formed via 1 mM UTP misincorporation by E. coli EcWT and EcH936A RNAPs (see scheme of the reactions in A). A black vertical line separates lanes originating from the same gel that were brought together.