The mechanism of transcription of T7 RNA polymerase shares some similarities to that of multisubunits RNA polymerases. As illustrated in Figure 12, T7 RNA polymerase first binds to a specific promoter sequence. Base-specific recognition of the promoter sequence involves the specificity loop that projects into the DNA binding cleft. After binding, unique groups of the polymerase promote melting of the double-stranded DNA leading to the formation and stabilization of the transcription bubble. The transition from duplex DNA in the binding region to open or melted DNA in the initiation region commences between the -5 and -4 positions, and involves an intercalating b-hairpin loop (Cheetham et. al., 1999b). The template strand is then led down into the active site by additional contacts with the surface of the enzyme, and de novo synthesis of RNA begins preferentially with a purine base.
During the early stages of transcription, T7 RNA polymerase forms an unstable initiation complex that synthesizes and releases transcripts that are 2-8 nucleotides in length (Figure 13). This phenomenon is known as the abortive phase of transcription; it is a general feature of all RNA polymerases. In the abortive phase, the contact with the binding region of the promoter are maintained while the leading edges of the initiation complex moves downstream, resulting in accumulation of DNA in the active site; this phenomenon is known as "scrunching" (Figure 14; Cheetham et. al., 1999b). The transformation to a stable elongation complex commences when the nascent RNA has achieved a length of about 9 nucleotides, but the transition is not completed until after 12-14 nucleotides have been synthesized (Figure 13). This transition is accompanied by the release of the upstream promoter contacts.
The elongation complex is stabilized by the thumb domain which wraps around bound template, thereby preventing the dissociation of the polymerase from the template while allowing for polymerase translocation (Bonner et. al., 1994). The next stage of the elongation stage is the exit of the RNA transcript through a surface channel. Based on the crystallographic analysis of the T7 RNA initiation complex, Cheetham et. al. proposed that the RNA transcript starts to peel off from the template strand when it only about 3 nucleotides in length. Further extension of the RNA-DNA hybrid would result in severe clashes with the T7 RNA polymerase N-terminal domain. It was also proposed an exit channel for the RNA transcript located between the polymerase thumb and N-terminal domains. This channel is shown in Figure 15 as a green arrow, and in Figure 16 by the small back arrow I.
Figure 14. DNA accumulation (scrunching) in the active site of T7 RNA polymerase. In (A), during initiation and before formation of the phosphodiester bond; in (B), after synthesis of pppGpGpG. Notice that ATP at position +4 is not reactive due to a methylene group at the bridging oxygen (Cheetham et. al., 1999b).
A more recent study using crosslinking methods by Temiakov et. al. demonstrated that in the elongation complex, RNA remains in an RNA-DNA hybrid until it reaches a length of about 8 nucleoties. Based on this work, it was also postulated that the RNA emerges to the surface of the enzyme when it is bout 12 nucleotide from the addition site. Interestingly, although the specificity loop interacts with the promoter in the initiation complex, it appears that it also interacts with the RNA product. It seems likely that association of the nascent RNA with the specificity loop facilitates disengagement from the promoter, and is an important part of the process that leads to a stable elongation complex (Temiakov, et. al., 2000).
Temiakov et. al. also demonstrated that two amino acids which are responsible for promoter recognition during transcription initiation interact with the nascent RNA product during the elongation phase. These residues are Gln758 and Arg746. To explain this result, it was postulated that during promoter clearance, the contacts between the specificity loop and the upstream-promoter DNA are broken and new contacts with RNA are established (Figure 16, see also Figure 17). Strikingly, Temiakov et. al.a also proposed a different exit channel for the RNA transcript based on molecular modeling studies using the available crystallographic data and crosslinking studies. This exit channel is located in the N-terminal domain, and is indicated in Figure 16 by the small black arrow II.
The inconsistency between the structure of the initiation complex obtained by x-ray crystallography and the one that emerged from biochemical studies could be explained if there is a large conformational change during the transition from an unstable initiation complex to a more stable elongation complex (see Severinov, 2001).
Based on the most recent biophysical and biochemical studies, a general catalytic mechanism of transcription initiation for T7 RNA polymerase can be proposed. The equation below describes this mechanism. The recognition of the promoter (P) by T7 RNA polymerase (R) leads to the formation of the closed complex (RPc), where k is the rate constant for the reaction step (see Figure 12).
The closed complex then isomerizes to an open complex (RPo), in which the DNA is partially melted. In the presence of ribonucleoside triphosphates, the RPo is ready to begin synthesis of RNA. The value of the association rate constant ( k1), the dissociation rate constant (k-1), and the dissociation constant (Kd), as been determined by stopped-flow kinetic assays are shown in Table 1 (Ujvari et.al., 1996). Similar kinetic determinations have found that transcription initiation is not limited by promoter DNA binding or melting but either to the first phosphodiester bond formation reaction leading to the synthesis of pppGpGpG or by a conformational change preceding that step (Jia et.al., 1997).
Recent x-ray crystallographic and biochemical studies have provided valuable information to formulate a catalytic mechanism for T7 RNA polymerase. In analogy to the two-metal-ion mechanism of polynucleotide polymerases proposed by Steitz (1998), a similar reaction mechanism can be written for T7 RNA polymerase (Figure 17). In this mechanism, the 3'-OH of the nascent RNA, which is base-paired to the DNA template, acts as the nucleophile (shown in blue). The incoming ribonucleoside triphosphate (shown in red), which is also base-paired to the DNA template, is positioned in the active site of the enzyme is such a way that its electrophilic a-prosphate is in close proximity both to the 3'-OH group of the growing RNA and to the two Mg(II) ions.
One of the Magnesium ions is coordinated to the 3'-OH group of the RNA, to one of the catalytic aspartate residues, and to a non-bridging oxygen of the a-phosphate. The main function of the metal ion A is to low the pKa of the 3'-OH group to facilitate the dissociation of the proton. Although this type of interaction might to some extend compromise the nucleophilicity of the 3'-OH group, the dissociation of the proton appears to be essential to promote the formation of the phosphodiester bond. On the other hand, the function of metal ion B, which is coordinated to the other aspartate residue and to one of the bridging oxygens, is to facilitate the release of the pyrophosphate group. Both cations also serve to stabilize the negative charges generated during the bipyramidal transition state.
Figure 17 also illustrates the role of Tyr639. It forms hydrogen-bonding interaction with the 2'-OH group of the incoming ribonucleoside triphosphate. Mutagenesis studies have demonstrated that the function of Tyr639 is to discriminate against deoxyriobose sugar. Thus, mutation of this amino acid to Phe will make T7 RNA polymerase a DNA polymerase (Sousa et. al., 1995). On the basis of x-ray crystallographic result obtained from the T7 RNA polymerase initiation complex, Cheetham et al (1999b) proposed that the role of His784 was to form hydrogen-bonding interaction with the 2'-OH group, and therefore, it was postulated that it also acted as a sugar discriminator. A posterior work describing the result of the His784 point mutant demonstrated that, alhough that mutation significantly reduces the activity of the polymerase, it does not significantly reduce the level of ribose discrimination (Brieba, et. al., 2000).