Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis

Abstract

In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites from different species appear to exhibit differential intrinsic transcript cleavage efficiency and have likely evolved to allow fine-tuning of the transcription process. Here we show that a single amino-acid substitution in the trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of intrinsic cleavage activity where the substituted tyrosine appears to participate in acid-base chemistry at alkaline pH for both intrinsic cleavage and nucleotidyl transfer. We extensively characterize this TL substitution for each of these reactions by examining the responses RNAP II enzymes to catalytic metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of the first phosphodiester bond formation by RNAP II requires wild type TL function and that H1085Y substitution within the TL compromises or alters RNAP II responsiveness to both TFIIB and TFIIF. Finally, Mn(2+) stimulation of H1085Y RNAP II reveals possible allosteric effects of TFIIB on the active center and cooperation between TFIIB and TFIIF.

Keywords: RNA synthesis; gene expression; general transcription factors; intrinsic cleavage; trigger loop.

Figure 1. A single amino acid substitution of tyrosine for histidine within the TL at Rpb1 position 1085 activates intrinsic RNA cleavage. (A) Scheme of transcription and cleavage reactions on a 5′-end immobilized template. Scaffold-assembled 5′ ³²P-labeled 9-mer RNA with purified RNAP II was advanced with ATP, CTP and GTP to the T33 stop through omission of UTP, resulting in a radiolabeled 32-mer. These stalled elongation complexes (EC32) were washed to remove NTPs, and then challenged with Mg²⁺ ions or buffer in the absence of divalent cations. (B) Gain-of-function intrinsic cleavage activity is specific to H1085Y and H1085Y/E1103G RNAP II enzymes. Arrows indicate the RNA cleavage products. Experiments shown are representative of at least two independent determinations.

Figure 2. Characteristics of the intrinsic RNA cleavage mediated by H1085Y RNAP II. Starting with RNAP II elongation complexes stalled at +32 (“EC32,” as in Figure 1), a schematic outline to the left of each experiment indicates, in minutes, the order of additions and washes (without metals) for that panel. (A) Requirement for divalent metal cofactors and the ability of H1085Y active center to resynthesize the excised RNA. H1085Y(EC32) was incubated in the absence or presence of catalytic divalent cations. “-Me²⁺” in the schematic indicates the absence of divalent metal cations in the wash buffer prior to readdition of divalent cations in each experiment as specified, followed by equilibration in buffer containing MgCl₂ with or without ATP for RNA re-synthesis. (B) Nucleotide sequence of the intrinsic cleavage template with the transcription stop highlighted (T33) and expected cleavage positions noted (A31, A30). (C) Determination of divalent cation preference for RNA cleavage (lanes 3–5) shows Mn²⁺ as the most potent cofactor at physiological pH (8.0). Re-synthesis of a 32-mer RNA with ATP only (lanes 6–8) indicates that 1- or 2- nt residues were excised (lane 4 vs. 7, panelB). (D) Dependence on alkaline pH. WT(EC32), H1085Y(EC32) and F1086Y(EC32) ECs were exposed to the basic range of the pH scale, from neutral (~7.0) to alkaline (~11), and intrinsic cleavage was quantified in (E), which shows a direct correlation between the efficiency of H1085Y-mediated cleavage and increasing pH (representative raw data shown in Fig. S2). Percent cleavage was determined by the amount of 31 nt RNA divided by the sum of 31/32 nt RNA. Note logarithmic scale for percent cleavage vs. pH. Error bars represent the range of two independent determinations or the standard deviation of three independent determinations. (F) TFIIS-supported hydrolysis of the second phosphodiester bond (P2) occurs concurrently and independently with the intrinsic cleavage of P1 with Mg²⁺. Outlined in F, the experiment in gel panel (G) shows a dose-dependent appearance of a 2 nt-shortened RNA at 10–100 nM TFIIS for WT and H1085Y, while the 1-nt shortened RNA derived from H1085Y ECs still persists at these TFIIS concentrations (lanes 13 and 14). Experiments are representative of at least two independent determinations.

Figure 3. An intact 3′-hydroxyl group on the ribose of a 3′-terminal UMP is essential for H1085Y-mediated intrinsic cleavage. (A) As outlined, RNAP II EC32 (elongation complex with 32-mer RNA) complexes were divided and their nascent RNAs were extended by 1 nt with either UTP, 3′-deoxy-UTP or 2′-deoxy-UTP. Washes omitting metal cofactors are delineated as “-Me²⁺.” Following a washing step in the absence of metal cofactors, ECs were then exposed to Mn²⁺ ions to initiate intrinsic cleavage where appropriate. (B) Comparison of cleavage reactions by WT (RNA lengths; 32, 33 nt) and H1085Y (RNA lengths; 32, 33 nt) RNAP II enzymes. A terminal 3′-dUMP prevented RNA hydrolysis by H1085Y RNAP II (lane 12), while the WT RNAP II control lanes reveal little WT RNAP II cleavage activity regardless of the nature of the 3′-NMP added. (C) Structures of the tested 3′-NMP modified sugars are depicted. Experiments are representative of at least two independent determinations.

Figure 4. H1085Y RNAP II ECs retain sensitivity to pyrophosphorolysis. (A) Set-up for pyrophosphorolysis of nascent RNA in RNAP II EC32 complexes in the presence of different divalent cations. Washes omitting metal cofactors are delineated as “-Me²⁺.” (B) “Me²⁺ treat” indicates RNAP II EC treatment for pyrophosphorolysis in the presence of specified divalent metal (Me²⁺) cations. Both WT and H1085Y RNAP II ECs are sensitive to pyrophosphorolysis, albeit H1085Y RNAP II shows lowered efficiency. Experiments are representative of at least two independent determinations.

Figure 5. Mn²⁺ restores the catalytic activity of H1085Y RNAP II in both abortive and productive initiation. (A) Scheme of transcription initiation on a 5′-immobilized pre-melted template onto which RNAP II is assembled. Positions of the transcription bubble and sequence features (TATA box) are numbered relative to the designated TSS (+1). GpG-primed, [α-³²P]ATP-extended 3-mer or a 12-mer RNA generated with NTPs including [α-³²P] radiolabeled ATP, are indicated. (B) Effects of divalent cations on the formation of the second phosphodiester bond in GpGpA (abortive initiation) or synthesis of a 12-mer RNA (productive initiation) were measured over time courses. “Me²⁺” indicates addition of divalent metal cations, which are specified under appropriate lanes of the gels shown. An asterisk marks abortive initiation products generated in productive initiation assays with Co²⁺ ions. Experiments are representative of at least two independent determinations.

Figure 6. Mn²⁺ restores the elongation activity of H1085Y RNAP II. (A) Outline of the RNAP II elongation assay, based on the 5′-end template immobilized scaffold assembly (Fig. 1A) with a ligated DNA fragment at its 3′-end to extend the RNA Run-Off (RO) product to a final length of 358 nt. Wash omitting metal cofactors is delineated as “-Me²⁺.” “t” represents time course of NTP addition in the presence of specified divalent metal cations. (B) Time-dependent elongation of a 10-mer RNA in the presence of divalent cations. Non-advanced ECs (10-mer) and ECs, stopped due to a portion of the original scaffold complexes failing to ligate the long run-off template (39-mer), are indicated. ³²P-labeled DNA marker (St.) to approximate RNA transcript lengths is included. (C) Average elongation rates in nucleotides per second were derived from two to three independent experiments, plotted in (Figure S4), where extension by 348 nts was used for calculations. Plus/minus values are the range of two determinations or the standard deviation of at least three determinations.

Figure 7. Mn²⁺ and TFIIB compensate for the deficiency of H1085Y in TFIIF stimulation of 3-mer RNA production. (A) The schematic timeline indicates, in minutes, the order of additions/washes for the experiment shown in B. WT and H1085Y RNAP II enzymes were preincubated with equimolar TFIIB or TFIIF, or TFIIB+TFIIF for 15 min at 25 °C. Following assembly on a bubble template, washed complexes were equilibrated in transcription buffer containing Mg²⁺ or Mn²⁺ ions and supplemented with a GpG dinucleotide primer and [α-³²P]ATP to initiate reactions for time points of 30 s and 2, 5, and 20 min. GpGpA products were quantified (Fig. S5) and the slope of linear regression lines calculated using GraphPad Prism 5.0d software. Wash omitting metal cofactors is delineated as “-Me²⁺.” (B) Plots represent the average relative rates of abortive initiation derived from linear regression with error bars indicating the range of two independent determinations, or the standard deviation of at least three determinations [No factors (black bars), TFIIB (white bars), TFIIF (gray bars), and TFIIB+TFIIF (blue bars)].

Figure 8. Trigger loop tyrosine at Rpo21/Rpb1 position 1085 is a candidate for acid-base catalysis during nucleotidyl transfer at alkaline pH. (A) RNAP II enzymes assembled on a IT-bubble nucleic acid scaffold were equilibrated in transcription buffer with pH 6.0, 8.0 and 9.5. Production of 3-mer RNAs by abortive initiation (3-mer) was monitored for 30 s, 2, 5, and 20 min time points. Average values from two to three experiments were plotted with the value for reaction at pH 8.0 set to 1 for each enzyme tested. A parallel experimental approach was always employed for comparison of transcription at pH 8.0 vs. 6.0 and for pH 8.0 vs. 9.5 for each enzyme. (B) In abortive initiation, H1085Y showed sensitivity to pH 6.0 and resistance to pH 9.5, while WT was resistant to pH 6.0 and sensitive to pH 9.5. H1085Q was partially sensitive to pH 6.0 and partially resistant to pH 9.5, relative to WT. Data are average of 2–3 independent replicates with error bars representing standard deviation (for three-replicate sets) or the range (for two-replicate sets).

Figure 9. In vivo effects of Mn²⁺ treatment and alteration of media pH on RNAP II TL mutants. (A) 10-fold serial dilutions of rpb1 TL WT, H1085Y and E1103G strains spotted onto media without or supplemented with increasing concentrations of MnCl₂ or MnCl₂ supplemented with MgCl₂. (B) Mild pH treatment on growth plates was accomplished by supplementation of standard media with 10 mM NaOH or 10 mM HCl or both. 10-fold serial dilutions of rpo21/rpb1 TL mutants and a control RPO21/RPB1 WT strain are shown.