Terminal adenylation in the synthesis of RNA by Q beta replicase

Proceedings of the National Academy of Sciences, 1975

Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qft replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous QI# replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Q# replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features.

Biochemistry, 1993

The existence of R N A enzymes that catalyze phosphodiester transfer reactions suggests that RNA-catalyzed R N A replication might be possible. Indeed, it has been shown that the Tetrahymena and sunY self-splicing introns will catalyze the template-directed ligation of R N A oligonucleotides (Doudna et al., 1989, 1991). We have sought to develop a more general R N A replication system in which arbitrary template sequences could be copied by the assembly of a limited set of short oligonucleotides. Here we examine the use of tetranucleotides as substrates for a primer-extension rea ion in which the 5'-nucleoside of the tetranucleotide is the leaving group, and the primer is extended by t he remaining three nucleotides. When the 5'-nucleoside is guanosine, the reaction is quite efficient, but a number of competing side reactions occur, in which inappropriate guanosine residues in the primer or the template occupy the guanosine binding site of the ribozyme. We have blocked these side reactions by using the guanosine analogue 2-aminopurine riboside (2AP) as the leaving group, in a reaction catalyzed by a mutant sunYribozyme that binds efficiently to 2AP and not to guanosine. We have begun to address the issue of the fidelity of the primer-extension reaction by measuring reaction rates with a number of different triplet/template combinations. Our results provide the basis for the further development of an RNA-catalyzed R N A replication system using short oligonucleotide substrates with novel leaving groups.

Journal of Molecular Biology, 1997

Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qb was studied utilizing a¯uorometric assay. A DNA oligonucleotide probe of sequence 5 H -d(TTTTTCC) was 5 H -end-labeled with pyrene. In this construct, the proximal thymine residues ef®ciently quench the¯uorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state¯uorescence increases by two orders of magnitude, the¯uorescence anisotropy increases, and a long¯uorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state¯uorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely re¯ects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qb replicase. The effect was utilized to access binding af®nities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16 C, equilibrium dissociation constants for different template midi-and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qb replicase during replication, exhibited discriminative af®nities, while their hybrid bound less ef®ciently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qb replicase does not require a 2 H hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A Á T base-pair, while that with cytosine is retained after Watson-Crick basepairing. These ®ndings can explain the af®nities of RNA-Qb replicase interactions reported here and in earlier investigations. The sensitivity of the described¯uorometric assay allows detection of RNA ampli®cation by Qb replicase in real-time.

Cell, 1978

Qp replicase polymerizes MDV-1 RNA at a markedly variable rate. Electrophoretic analyses of partially synthesized strands showed that a few of the elongation intermediates are much more abundant than others, reflecting a variable rate of chain elongation.

European Journal of Biochemistry, 1989

We consider a template-instructed MDV-1 RNA replication catalyzed by Qp replicase. The relaxation of the effective binding of the growing chain to the enzyme is simulated, considering a Markov process where refolding events in the chain are concomitant with chain elongation events. When realistic transition probabilities are considered, it becomes evident that the pause sites in replication are due to relaxation of the binding by folding of the growing chain.

European Journal of Biochemistry, 1972

The largest polypeptide obtained as a product of Qj3 RNA-stimulated cell-free protein synthesis is the replicase (R) protein. The R polypeptide synthesized in vitro has a chain length of approximately 550 residues and is identical in size to the @ chain of purified replicase. From its electrophoretic behavior a t pH 8.5, a net charge of -27 and a cysteine content of > 15 residues can be deduced. A portion of R polypeptide synthesized in vitro binds to added Qj3 RNA and this binding is more resistant to increased ionic strength than the binding of the positively charged coat protein to Q@ RNA. Another portion of the R polypeptide binds to some constituent of the Escherichia coli extract to form a 20-5 complex.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1996

Four 25mer oligonucleotides containing m4T (T * > with the 3' + 5' sequences AT *-A, AT *-C, G-T I-G and G-T *-C, together with four oligonucleotides containing the same sequences with unmodified T, were studied for the effect of the immediate 5' and 3' bases flanking m4T on in vitro replication. Under enzyme-limiting conditions, all m4T-containing oligomers showed a varying sequence-dependent blockage at the base 3' to m4T. However, with time and high concentrations of dNTP's, full replication beyond m4T did occur with Klenow fragment (Kf), although substantial blockage remained, particularly in the case when the sequence was 3'-AT *-A-5'. The extent of blockage at the base 3' to m4T, compared to complete replication using Kf, followed the order AT *-A > AT *-C 3 G-T *-C 2 G-T *-G. Two other DNA polymerases, HIV-RT and Sequenase, which, unlike Kf, do not have 3' proofreading activity differ, agreed with data obtained with Kf. We conclude that the extent of complete replication of m4T-containing oligomers is primarily a function of the 3' flanking base with a lesser effect of the 5' base and requires specific dGTP insertion opposite m4T. These data suggest that a 5' G C pair favors the formation of the following T *. G and this latter step is rate-limiting. However, once a T *. G pair is formed, rapid elongation to full length transcripts occurs.

Journal of Molecular Biology, 1998

An internal site on bacteriophage Qb RNA, the M-site (map position 2545 to 2867), was recently shown by us to be required for the ef®cient initiation of minus strand synthesis by Qb replicase. In a more detailed mutational analysis, we show here that the essential elements within the M-site consist of two successive stem ± loop structures followed by a bulge loop of unpaired purines, located at nucleotides 2696 to 2754 on the tip of a long, imperfectly base-paired stalk. Mutational changes affecting the sequences of paired or unpaired nucleotides in this segment reduced the template ef®ciency only mildly. The only severe effects were observed when one of the helical stems or the unpaired bulge was completely deleted or substantially shortened. We conclude that the threedimensional backbone arrangement of these three elements constitutes the feature recognized by replicase. The role of the long stalk remains undetermined, because mutations that either stabilized or disrupted its base-pairing barely affected template activity, and even deletion of a major portion of one of its strands did not cause complete inactivation. Earlier evidence had implicated protein S1 (the a subunit of replicase) as the mediator of the M-site interaction. The lack of an active M-site on the Qb RNA template has the same quantitative and qualitative effects on template recognition as the absence of the S1 protein from replicase in the presence of wild-type RNA. We therefore believe that the M-site interaction explains most of the role of S1 protein in the replication of Qb RNA by replicase.

Proceedings of the National Academy of Sciences, 1976

Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured ...

European Journal of …, 1995