The hairpin ribozyme catalyzes a reversible phosphodiester cleavage reaction. We examined the roles of conserved nucleobases in catalysis using an abasic ribozyme rescue strategy. Loss of the active site G8 nucleobase reduced the cleavage rate constant by 350-fold while loss of A9 and A10 nucleobases reduced activity less than 10-fold. Certain heterocyclic amines restored partial activity when provided in solution to the variant lacking G8. Heterocyclic amines that were capable of rescue shared the exocyclic amine and cyclic amide in common with the Watson-Crick hydrogen bonding face of guanine. In contrast to the shallow pH dependence of unmodified ribozyme activity, rescue activity increased sharply with decreasing pH. These results support a novel model for RNA catalysis in which a cationic nucleobase contributes electrostatic stabilization to negative charge developing in the transition state.

Unlike DNA polymerases, an RNA polymerase must initiate transcription de novo, that is binding of the initiating (؉1) nucleoside triphosphate must be achieved without benefit of the cooperative binding energetics of an associated primer. Since a single Watson-Crick base pair is not stable in solution, RNA polymerases might be expected to provide additional stabilizing interactions to facilitate binding and positioning of the initiating (priming) nucleoside triphosphate at position ؉1. Consistent with base-specific stabilizing interactions, of the 17 T7 RNA polymerase promoters in the phage genome, 15 begin with guanine. In this work, we demonstrate that the purine N-7 is important in the utilization of the initial substrate GTP. The fact that on a template encoding AG as the first two bases in the transcript (as in the remaining two of the T7 genome) transcription starts predominantly (but not exclusively) at the G at position ؉2 additionally implicates the purine O-6 as an important recognition element in the major groove. Finally, results suggest that these interactions serve primarily to position the initiating base in the active site. It is proposed that T7 RNA polymerase interacts directly with the Hoogsteen side of the initial priming GTP (most likely via an interaction with an arginine side chain in the protein) to provide the extra stability required at this unique step in transcription.The initiation of transcription imposes some unique mechanistic requirements on an RNA polymerase. In contrast to events occurring during elongation, at the initial step of transcription initiation, two substrate nucleoside triphosphate molecules must position accurately in the active site. Clearly a part of the binding energetics is derived from Watson-Crick interactions between the incoming bases and those in the template strand of DNA, but just as clearly, base pairing interactions are not sufficient to provide the binding energetics required for full function. Indeed, a single Watson-Crick base pair is unstable in solution (1).It is understood that for the Watson-Crick placement of the elongating nucleotide (position ϩ2 at initiation), additional energy for binding of the nucleotide comes from interactions between its triphosphate, magnesium, and protein functional groups (2-5). This interaction would not be expected to be important in binding the initiating (ϩ1) nucleotide, and it has been shown in the T7 system that guanosine monophosphate and even the nucleoside guanosine have K m values comparable to or lower than that of the triphosphate GTP (6). Some additional interaction(s) must be at play.Most RNA polymerases show some preference for the initial base of the transcript. Escherichia coli RNA polymerase promoters often initiate with ATP, although some promoters begin with other NTPs at the first position in the transcript (7,8). Of the 17 phage RNA polymerase promoters in the T7 genome, the canonical ϩ1 position of 15 begins with GTP, while two promoters begin with ATP (9). Recent studies have demonstrated that ...

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T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA., T7 RNA polymerase will synthesize a 'ladder' of poly-G RNA products. At each step, the ratio of elongation to product release is consistently approximately 0.75 until the RNA reaches a length of approximately 13-14 nt, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore becomes sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7-deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination.

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