RNA replication by Q beta replicase: a working model.

Abstract

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Qj8 replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I-and site 1I-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Qf3 replicase.Q03 replicase is the RNA-dependent RNA polymerase responsible for replicating the single-stranded RNA genome of the coliphage Q0 (1). The replicase is a heterotetramer comprising a virally encoded subunit (2, 3) plus three host proteins: ribosomal protein S1 (4, 5) and elongation factors Tu (EF-Tu) and Ts (6). Q03 replicase is remarkable in three respects: (i) it effects a 10,000-fold amplification of the 4200-nucleotide single-stranded RNA of Q0( during the very short infection time of the phage (7), (ii) it specifically replicates the viral genomic RNA in the presence of a vast excess of host RNA, and (iii) it copies entire template RNAs, from 3' terminus to 5' terminus, without utilizing endogenous primers.The first feat is accomplished by exponential amplification (8). The polymerase uses the plus strand of the phage genome as a template to produce a complementary RNA, referred to as the minus strand. Both the plus and minus strands serve as templates for the replicase, thus the amount of phage RNA doubles with each replication cycle. The molecular mechanism by which the enzyme achieves template recognition and initiates complementary strand synthesis at the 3' end of the RNA, however, has yet to be resolved. In a previous report (9), SELEX (systematic evolution of ligands by exponential enrichment) was used to generate two families of RNA ligands (referred to as class I and class II) that bound Q0 replicase with nanomolar dissocation constants.The class I ligands possessed single-stranded regions containing A's and C's that were essential for binding the replicase. A region of the plus strand of the Q0( genome that is required for replication, and that binds specifically to Q0( replicase, possesses a similar sequence motif (10, 11). The class II RNAs had polypyrimidine tracts that were required for high affinity binding to the replicase. The Q03 genomic minus strand, as well as most of the small replicated RNAs identified by in vitro experiments, possess similar pyrimidine rich regions (9). Importantly, the two classes of RNAs did not compete for binding to the replicase, indicating that two independent binding sites exist on the phage polymerase. The two regions of Q03 replicase that ar...

Structure of the Qβ replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins

“…Qβ replicase is the most efficient in vitro system for nucleic acid amplification, far more efficient than PCR or isothermal amplification systems (17). However, the utilization of Qβ replicase for amplification of desired sequences has yet to be realized because of its puzzling and extraordinary template specificity requiring neither promoters nor primers (7,8). Although the described structure does not provide an immediate solution to this problem, it paves a way for a rational approach towards reaching this goal.…”

“…EF-Ts may stabilize EF-Tu in a conformation with affinity for Qβ RNA templates instead of aa-tRNAs (9). Ribosomal protein S1 mediates recognition of the Qβ plus strand (8), whereas the RNA chaperone Hfq is believed to mediate the access of the β-subunit to the 3′ end of the plus strand (10).…”

“…Subsequently, EF-Ts catalyzes the exchange of GDP for GTP on EF-Tu. In the Qβ replicase, the EF-Tu:EF-Ts subcomplex is necessary for initiation of replication of both the minus and plus strands (7) with EF-Tu being involved in template binding and recognition (8). EF-Ts may stabilize EF-Tu in a conformation with affinity for Qβ RNA templates instead of aa-tRNAs (9).…”

pTAC2, -6, and -12 Are Components of the Transcriptionally Active Plastid Chromosome That Are Required for Plastid Gene Expression

“…Oleskina et al (1999) also identified ribosomal proteins that are associated with plastid DNA. Studies on phage Q replication demonstrated that S1 and the elongation factors EF-TU and EF-TS are also subunits of the b-RNA replicase (Brown and Gold, 1996). Likewise, EF-Tu, S3, L12, and L29 could have a dual function in plastid gene expression in that they coordinate replication, transcription, and translation.…”

Autonomous Role of 3′-Terminal CCCA in Directing Transcription of RNAs by Qβ Replicase

“…The interspersed U residues were designed to prevent the replicase slippage anticipated on longer A tracts. As well as testing a role for C clusters as nonspecific transcriptional enhancers, these RNAs permitted testing a role for a short C cluster at an appropriate spacing upstream of the 3Ј-initiation box in enhancing transcription (proposed in reference 18), perhaps by binding to so-called Site II of the EF-Tu subunit (8).…”