Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase

discriminating role similar to that of Asn458 in the insertion site. Alternatively, upon the substrate binding to the insertion site the structural elements that we are now assigning to the pre-insertion site might move towards the RNAP active center to constitute a single "closed" insertion site. We are currently dissecting the roles of residues in the pre-insertion site of the bacterial RNAP in selection of sugar and the base moieties of the incoming substrate.


INTRODUCTION
Copying (replication) and read-out (transcription) of genetic information contained within cellular genomes are carried out by an array of DNAPs and RNAPs, respectively. These enzymes operate within the same compartment, accessed by both types of substrates, r-and dNTPs, and control their fidelity at both co-and post-synthetic steps in order to avoid the so-called 'error catastrophe', when the amount of mistakes exceeds redundancy of the system. The first mechanism relies on the discrimination against the "wrong" nucleotide, whereas the second mechanism is invoked after the incorporation of a mismatched nucleotide.
The initial selection of the cognate substrate is comprised of two parts: the selection of the nucleotide complementary to the template DNA base and the selection of the correct sugar.
The mechanism for sugar selection has been extensively studied in several DNAPs and in the single-subunit T7 RNAP. DNAPs actively discriminate against the rNTP via a "steric gate" formed by the Glu and Phe side chains (1-5), which sandwich the substrate sugar moiety and exclude the 2'-OH, while the substrate is positioned in the insertion site that has "closed" (active) configuration. In single-subunit T7 RNAP, Tyr639 hydroxyl has been implicated in the positive selection of the ribose via formation of a hydrogen with the 2'-OH group (6)(7)(8). In contrast to DNAPs, T7 RNAP commences the substrate selection in the inactive "open" conformation, while the substrate and the Tyr639 side chain are located in the so called pre-insertion site far away 4 yet carry out the transcription cycle in a nearly identical manner and could utilize analogous structural elements during catalysis (12)(13)(14). These similarities suggest that the basic substrate selection mechanism might also be conserved, and that a residue analogous to the Tyr639 would play a critical role in maintaining the ribose specificity in multi-subunit RNAPs. However, modeling of the substrate NTP bound to the T. thermophilus RNAP active site (15) suggests that β' Asn458 (E. coli numbering is used throughout) within a highly conserved sequence motif N 458 ADFDGD 464 that includes the catalytic Asp triad (β'Asp460, 462, 464) (16-18) could mediate specific recognition of the O2' ribose atom (Fig. 1A). Thus, Asn458 is likely not a structural analog of Tyr639, as the latter initially recognizes the ribose in the pre-insertion site.
To evaluate the role of β' Asn458 in substrate selection by a bacterial RNAP we prepared and tested the conservative substitutions of this residue for their effects on utilization of the noncognate substrates by the E. coli RNAP. Here we demonstrate that, in accordance with the structural predictions, these substitutions lead to a loss of discrimination of sugar moiety.

EXPERIMENTAL PROCEDURES
Reagents -All general reagents were obtained from Sigma and Fisher; rNTPs and dNTPs from Amersham, PCR reagents from Eppendorf, and restriction and modification enzymes from NEB. Oligonucleotides were obtained from Integrated DNA Technologies, [α 32 P]-NTPs from NEN. Plasmid DNAs and PCR products were purified using spin kits from Qiagen.
Construction and purification of altered RNAPs -The E. coli rpoC gene was subjected to site-directed mutagenesis with two fully complementary oligonucleotides that defined the desired mutation. The shortest corresponding restriction fragment of the rpoC gene was completely sequenced at Genewiz Inc., and transferred into the overexpression vector pIA299, which encodes the rpoA-rpoB-rpoC gene cassette under the control of T7 promoter. The wild-type and altered RNAPs were expressed and purified as described previously (19).
In Vitro Transcription Reactions -All templates for transcription reactions were generated by PCR amplification. Plasmid pIA171 encodes a 29-nt U-less transcribed region under control of T7A1 promoter followed by a his pause site (20). Plasmid pIA349 encodes a 37-nt U-less

RESULTS
Experimental set-up -To determine whether β'Asn458 residue is critical for the interaction with the 2' OH, we changed Asn458 to Asp and Ser; these are the conservative changes that would not be expected to substantially alter the structure of the protein but would change either the chemical properties (Asp) or the size (Ser) of the discriminating Asn side chain. The altered enzymes were overexpressed from a polycistronic vector that allows assembly of the core α 2 ββ' RNAP in vivo (19), purified, and tested for the ability to discriminate between the cognate and non-cognate substrates. Similarly purified wild-type (WT) RNAP was used as a control.
We assembled transcription elongation complexes (TECs) on pIA349 template that encodes a T7A1 promoter (21). On this template, TECs can be initially halted at position 37 by withholding UTP from the reaction mix ( Fig. 2A), purified by gel-filtration to remove the unincorporated substrates, and then "walked" to the next template position during addition of a subset of NTPs. This approach allows to test whether RNAP will efficiently incorporate a given substrate, and whether it will extend the nascent RNA transcript after a misincorporation event.
We used a similar approach to measure rC/dC selectivity using pIA171 template (20) instead.
Substitution of Asn458 leads to increased dNTP utilization -We formed a halted TEC on an appropriate template and tested extension of 32 P-labeled RNA upon addition of different unlabeled substrates. For each r/dNTP pair assayed, selected substrate was added to a concentration ranging between 0.1-62.5 µM for NTPs and 2-1250 µM for dNTPs, reactions were allowed to proceed for 2 min at 37 o C, and quenched with STOP buffer (Fig. 2B).
As can be seen from representative gel images (Fig. 2B), the WT RNAP exhibits strong preference towards rNTP substrates, while in the N458D variant these preferences are relaxed.
For example, a 2-min incubation with 0.1 µM rGTP led to efficient extension of the nascent RNA by the WT RNAP, whereas ~100-fold higher concentration of dGTP was required to achieve the same degree of extension. For the purpose of quantitative comparison we selected the ratio between concentrations of dNTP and rNTP, when half of the transcripts are extended by one nucleotide, as the discrimination quotient. The selectivity of incorporation of different r/d NTP pairs by WT RNAP ranges from 120-to 1200-fold ( Fig. 2C) suggesting that the substrate selection depends not only on the identity of the sugar moiety but also on the base structure, even when the Watson-Crick base pairing is maintained. Similar variations have been reported for T7 RNAP (6) and DNAP I (2); in the latter case selectivity correlates with the strength of base pairing, consistent with the proposed mechanism to selectively stabilize incoming A and T substrates to balance their weaker interaction with the template base (22). The same could also be true for the E. coli RNAP as discrimination capacity generally increases with the weakening of the base pair except for rU/dT selection, which could be additionally influenced by the presence of the 5-methyl group on thymine.
As expected, substitution of Asn458 for Asp led to substantially relaxed sugar discrimination ranging from 4.8-fold loss of discrimination for rG/dG pair to 26-fold loss in case of rU/dT pair. Substitution of Asn458 for Ser led to a smaller loss of preference for the ribose, whereas substitution of an adjacent Tyr457 residue did not alter sugar selection (Fig. 2C). The maximal observed effect for N458D RNAP was somewhat less than that reported for the T7 Y639F RNAP (6,8); the base-specific order of r/dNTP selectivity was also different between the bacterial and T7 enzymes ( Fig. 2C and ref(6)). The direct comparison of these data is not straightforward, however, as we have used pre-steady state assays of substrate incorporation by the TEC, whereas Sousa et al used multi-round assays that include both the initiation and elongation steps, which resulted in different discrimination efficiencies for the same RNAP depending on the assay design (6,8). In T7 RNAP Tyr to Phe substitution likely completely disrupts polar discriminative interactions with the substrate ribose, whereas Asn for Asp and Ser substitutions in the bacterial enzyme might still maintain specific hydrogen bonding with rNTP sugar moiety. In addition, Asn458 might be not the only residue participating in the ribose recognition in the active site. Indeed, according to the modeling guanidinium group of a highly conserved in bacteria and eukaryotes β'Arg425 appears proximal to the substrate sugar (Fig. 1B).
The experiments are now underway to test the role of β'Arg425 in substrate selection.
Molecular modeling of the multi-subunit enzyme pre-insertion site (10) suggested that E. coli β'Thr790 residue could play a role in ribose selection analogous to that of the T7 Y639 residue. However, the T790V RNAP not only did not loose preference for rNTP substrates, it actually exhibited more stringency in substrate selection relative to the WT, ranging from 1.1fold effect in case of rC/dC to 4.8-fold effect for rU/dT (Fig. 2C). These data underscore the importance of Thr790 residue in substrate selection but argue against involvement of its hydroxyl group in positive selection of the substrate ribose; less conservative substitutions of Thr to Ala and Leu led to gross defects in catalysis (data not shown).
A greater loss of discrimination upon the Asn to Asp substitution suggests that charge distribution on the discriminating protein group is more important than the side chain size. In the absence of the experimental crystallographic data on the RNAP/substrate complex structure we propose a model that is certainly too tentative to predict the exact scheme of the hydrogenbonding with the substrate but nonetheless allows to zero in on the three protein groups that likely interact with the substrate ribose: Asn458 side chain, Asn458 main chain carbonyl oxygen, and Arg425 guanidinium group. In the high-resolution crystal structure of the T. thermophilus RNAP holoenzyme (17) these three groups form internal hydrogen bonding network (Fig. 1B).
Interestingly, the main chain conformation of Asn458 does not fall in the most favorable region on the Ramachandran plot (φ=-68 o ;ψ=77 o ). The stabilization of this unfavorable conformation likely comes from the hydrogen bonding of the Asn458 main chain carbonyl with Arg425 and with its own side chain (Fig. 1B). The latter interaction would prevent flipping of the Asn458 side chain amido group that might be crucial for the proper sensing of the substrate ribose. This network of interaction would be enhanced upon binding of the rNTP substrate. In the model, Arg425 as well as the main chain carbonyl oxygen and the side chain amide of Asn458 make contacts with the O3' group, whereas the Asn458 side chain oxygen likely recognizes O2' group of the substrate ribose. The modeling of N458S substitution showed that though the interactions with the sugar would be weakened due to the smaller size of the Ser side chain, its hydroxyl group may preserve the framework of protein-protein and protein-substrate contacts similar to that of Asn (data not shown). In contrast, the negatively charged Asp side chain would lack the interactions with the main chain carbonyl potentially affecting both the main chain and side chain conformations and thus distorting the optimal orientation of the discriminating residue. In addition, the acidic Asp458 side chain might also form a salt bridge with the adjacent Arg425 that would further perturb the Asp interactions with the substrate ribose.

Substitution of Asn458 leads to profound defects in transcript elongation -In addition to
being instrumental for the elucidation of the molecular mechanisms of substrate selection, RNAP variants with altered substrate selection properties could be used to determine the contribution of individual transcript bases to recognition of regulatory signals. This approach is particularly important for the functional analysis of transient kinetic intermediates, such as those occurring during transcription termination, when artificial stalling of complexes leads to trapping of the off-pathway species (23), and relies on the ability of nucleotide analogs to affect RNAP response to a particular signal (nucleotide analogs interference mapping; NAIM). This analysis requires that RNAP is able to incorporate substrate analogs bearing modifications at various positions (24). For example, T7 RNAP with double substitution Y639F/H784A has been used to demonstrate the effect of replacement of individual residues in the nascent RNA with dNTP analogs on termination (24). However, to apply this strategy one needs to ascertain that the mutation in RNAP, while allowing incorporation of unusual substrates, does not lead to a defect in recognition of the transcription signals intended for study.
We wanted to test if the N458D RNAP could prove similarly useful in mapping the requirements in the nascent transcript during elongation/termination. We studied the elongation properties of the N458D RNAP on pIA349 template that encodes several well-characterized pause sites (Fig. 3). We found that N458D substitution confers a strong elongation defect: the rate of transcription elongation was dramatically reduced (more than 20-fold), and the enzyme paused strongly early in the transcribed sequence. We conclude that N458D RNAP, albeit able to efficiently incorporate dNTPs into the nascent RNA, is not suitable for NAIM due to its profound elongation defects. N458S RNAP also displayed a reduced elongation rate but the defect was less pronounced (5-fold, Fig. 3 and data not shown). These results are also in a good agreement with our modeling. In the RNAP holoenzyme structure β'Arg425 makes strong hydrogen bonds with β'Asp464 from the catalytic triad. We presume that these interactions are crucial for the proper positioning of the Asp464 side chain that is likely required to optimize the coordination of the major catalytic Mg 2+ ion (cMg1, Fig. 1B). In N458D enzyme side chains of

DISCUSSION
The major conclusion of this work is that β'Asn458 residue in the E. coli RNAP provides the recognition of sugar moiety on the incoming NTP substrate. No significant changes in misincorporation of non-templated NTP substrates arose from Asn458 substitutions (see Supplemental Data), indicating that the determinants for the sugar and base selection are nonoverlapping, as was observed for the single-subunit T7 RNAP (11). The proposed ribose selection mechanism would be also likely valid for eukaryotic RNAPs that contain a highly conserved counterpart of β'Asn458; indeed this role for the corresponding Asn479 residue in Rpb1 had been proposed earlier (14).

For both T7 and E. coli RNAPs the selectivity is far below the values reported for the
DNAPs that exclude the rNTP substrates with several thousand-to a million-fold efficiency (1,2). These differences could be explained, on one hand, by the fact that the levels of rNTPs are at least 10-fold higher in the cell than the levels of the corresponding dNTPs (25). Thus, to prevent incorporation of rNTPs that could prove to be lethal (26), DNAPs must impose the strict discrimination mechanism, which is achieved by the steric exclusion of the ribose 2' hydroxyl.
On the other hand, RNAPs might face the opposite problem: the relatively inefficient (e.g. via a single H bond) discrimination between r and dNTPs could result not only in the synthesis of compromised messages but also in "draining" of the dNTPs pool. Cellular RNAPs would be expected to evolve tighter control mechanisms as compared to the phage ones since at the time phage RNAP becomes engaged in transcription the host cell is usually moribund.
In DNAPs, the substrate recognition is thought to occur exclusively in the insertion site (27). In contrast, in the T7 TECs substrate can be bound in either the pre-insertion or in the insertion sites located 10 Å apart, suggesting that RNAPs may select substrates in two rather than in a single site (9,10). The presence of a pre-insertion site in which the substrate can be "sampled" prior to catalysis was also proposed recently for multi-subunit RNAPs (10,28). Our present data on Asn458, which is adjacent to the active site, suggest that in bacterial RNAPs substrate selection, at least in part of rNTP/dNTP selection, occurs in the insertion site. Thus the sugar selection may principally occur in the pre-insertion site in T7 but in the insertion site in bacterial RNAPs. If discrimination in favor of rNTP binding were to occur predominantly in the insertion site, persistence of these interactions after catalysis might hinder the movement of the incorporated nucleotide from the n to (n-1) site, thereby slowing translocation and the rate of polymerization. In T7 RNAP, where the pre-insertion site binding seems to be preferable, the Tyr639-2'OH contact might be compromised during the transition to the closed form. This might explain a faster rate of T7 RNAP transcription as compared to the multi-subunit cellular enzymes (29) which, facing more regulatory and fidelity constraints, might have evolved somewhat different mechanisms for substrate selection.
One cannot, however, rule out the possibility that rNTP/dNTP discrimination occurs both in pre-insertion and insertion sites by the different sets of residues. Indeed, substitution of the E. coli β'Thr790 that is located in the bridge helix and likely belongs to the putative pre-insertion site for Val not only did not decrease the enzyme preference for the ribose, but instead increased discrimination up to ~5-fold (Fig. 2C), implying the direct interactions of β'Thr790 (or an adjacent) residue with the substrate. Since β'Thr790 is located 18Å away from the active (insertion) site and ~8Å from the modeled substrate ribose, this result provides strong support to the hypothesis of existence of the substrate pre-insertion site in multi-subunit RNAPs. It is therefore possible that some other residue from the pre-insertion site may play a ribose 15 discriminating role similar to that of Asn458 in the insertion site. Alternatively, upon the substrate binding to the insertion site the structural elements that we are now assigning to the pre-insertion site might move towards the RNAP active center to constitute a single "closed" insertion site. We are currently dissecting the roles of residues in the pre-insertion site of the bacterial RNAP in selection of sugar and the base moieties of the incoming substrate.

Substitution of Asn458 does not alter misincorporation
The recognition of the cognate substrate relies on selection of both the correct sugar moiety and the complementary base. To analyze the miscoding properties of N458D RNAP we have utilized a similar approach where we added a large excess of a noncomplementary rNTP substrate and monitored the RNA chain extension by the WT and altered enzymes ( Fig S1A). As expected, RNAP incorporates a wrong base very reluctantly; however, no notable differences were observed among the three enzymes.
This result is reminiscent of the observations made for the T7 Y639F RNAP and phage φ29 Y254V DNAP, which display a relaxed sugar discrimination but have no defects in base recognition (1,2).

Substitution of Asn458 does not alter extension of the dNTP primer
Upon addition of the wrong substrate nucleotide, RNAP could also fail to extend the misincorporated transcript efficiently and edit it through the preferential cleavage of the compromised 3' end fragment. This mechanism was proposed to contribute to fidelity of transcription in both bacterial and eukaryotic systems (3,4). We tested if the substitution of β'Asn458 would alter the efficiency of the extension of 3' deoxynucleotide ( Fig S1B).
Again, the extension efficiency of misicorporated dNMP did not differ significantly between the WT and mutant enzymes.