RNA replicase
Qβ replicase


Biebricher, C.K., and Gardiner, W.G. Molecular evolution of RNA in vitro. Biophysical Chem. 66, 179-92 (1997)
Blumenthal T, Carmichael G.G., RNA replication: function and structure of Qβ -replicase.Ann. Rev. Biochem. 48:525-48, 1979 - available online.
Brown, D. and Gold, L. RNA replication by Qβ replicase: A working model PNAS 93, 11558-62 (1996)
Chetverin, A.B., and Spirin, A.S. Replicable RNA vectors: prospects for cell-free gene amplification, expression and cloning. Prog. Nucl. Acid Res. 51, 225-70 (1995)
Konarska, M.M., and Sharp, P.A. Replication of RNA by the DNA-dependent RNA polymerase of phage T7. Cell 57, 423-31 (1989)
Su, Q., Schuppli, D., Tsui, H.T., Winkler, M.E., and Weber, H. bly reduced Qβ replication, but normal phage MS2 replication in an E. coli K12 mutant with inactivated Qβ host factor (hfq) gene. Virology 227, 211-14 (1997)
Wettich A., Biebricher C.K. RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli. Biochem 40, 3308-3315 (2001) available online.

I- Qβ replicase

1- "Replicase" - makes copies of template (through (-) strand intermediate)
2- Autocatalytic synthesis (product is also a template)
3- Qβ RNA is the only effective natural template
4- (+), (-) strands, but not dsRNA are templates
5- (-) strand is best template
6- Host factor required for (+) strand as a template (leviviruses don't need hf)
7- Release of product is rate limiting step (enzyme binds tightly)
II- Polypeptide composition - 4 polypeptides

1- S1 - ribosomal protein (plus strand RNA binding site)
2- Tu, Ts - host protein synthesis chain elongation factors
III- Template specificity

1- Qβ RNA is a good template, levivirus RNAs are poor templates
2- Levivirus replicases are unstable, but don't use Qβ RNA as a template and don't require hf (host factor)
3- hf required to express several E.coli stationary phase genes
4- Qβ selected to grow on hf- E.coli is mutated at 3' end
IV- In vitro Darwinian selection

1- Serial transfer into excess enzyme (with substrates) yields small templates
2- Small RNAs (variants) are better templates than viral RNA
3- Variants have lots of secondary structure
4- Can select variants (e.g. EtBr resistance)
V- Variant RNAs as templates for Qβ replicase

1- Variant replication requires neither hf nor replicase subunit S1
2- Sequences w/o secondary structure are poor templates (form dsRNA)
3- Can enzyme generate its own template? Poisson dilutions
4- Some templates are recombinants (can include host sequences)
5- Minivariants are similar to known sequences
6- Normal infection produces small variants, but they are poorly encapsidated
8- Do plant virus need to avoid generating "parasitic" templates?
9- "Parasitic" small RNAs can infect T7 DNA dependent RNA polymerase

VI- In vivo control of RNA synthesis

1- Ribosomes compete with replicase for binding
2- (+) strand is sequestered by encapsidation
3- Limiting S1, hf may prevent use of plus strand as a template