Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA.

Abstract

It has been proposed that the nucleotide sequences of the 3' terminal extracistronic regions of phage RNA plus and minus strands have been strictly conserved during evolution because they are stringently required for recognition by the viral replicase. We The 5'-and 3'-terminal regions of Q13 RNA comprise nontranslatable sequences of 61 and >61 nucleotides, respectively (1-3). A similar situation obtains in the case of the RNA of the group I phages MS2, f2 and R17 (4-7). Although about 3% of the nucleotide positions in the coat cistron differ from one group I RNA to another (7-9), the terminal extracistronic regions, comprising more than 150 nucleotides, are invariant. This observation led to the proposal that the replication of these phages depends strictly on the primary structure of the extracistronic sequences (9); in particular it was suggested that the specific interaction of phage RNA with its cognate replicase is mediated by these regions (6) Mmol of Tris-HCl (pH 7.5), 4 Mmol of KCl, and 20 nmol of EDTA were added, giving a final volume of 20 A. After 15 min at 370, 10 IA were transferred to 10 M1 of a mixture of the same composition but containing no RNA template. After 15 min at 370, this procedure was repeated, for a total of nine transfers. Ten microliters of the 10th incubation and 2 units of replicase were added to 20 Al of fresh reaction mixture and incubation was continued for 30 min. Ten microliters of fresh mixture and 4 units of replicase were added, and after an additional 30 min another 30 pl of mixture were added and incubation was continued for a further 90 min. After extraction with an equal volume of phenol at 20°the aqueous layer was filtered through a column (7-mm diameter) of Sephadex G-50 (7 cm) and Chelex-100 Na+ (0.5 cm). The pooled excluded fractions were adjusted to 0.05 M Tris * HCl (pH 7.5), 0.1 M NaCl, 5 mM EDTA, and the RNA was precipitated with 2 volumes of ethanol at -10°and recovered by centrifugation (10,000 rpm in a Sorvall HB-4 rotor, 10 min at -10°).The RNA was dissolved in 150 MAl of 0.5 mM EDTA and centrifuged through .a 5-23% sucrose gradient in 50 mMTris-HCl (pH 7.5)-5 mM EDTA. The fractions containing 30S RNA were pooled and precipitated with ethanol and the RNA was dissolved in 20 Ml of water. The yield of RNA was 4 Mg. Two micrograms of this product were denatured and used as template to initiate a series of nine further transfers, as described above. A 2-Ml sample of each of the incubations (except for the 1st, 5th, 10th, and 21st) was taken to determine acid-insoluble radioactivity.Serial replication of wild-type Q# RNA (extracted from phage) was carried out in a similar fashion, except that incuba-

Single nucleotide variations: Biological impact and theoretical interpretation

“…Rather than using laborious random mutagenesis and screening to identify functional residues,144,145 site-directed mutagenesis studies146 may be efficiently guided by computational predictions with high rates of success 134,147–149. Besides selecting strongly deleterious mutations that knock out protein function, often it is desirable to select mutations with an intermediate impact in order to redirect the protein activity 135.…”

Transfection of Enterobacteriaceae and its applications.

“…In the field of procaryotic molecular biology, the area of directed mutation in vitro (83, 114,115,172) is likely to receive continued attention; physical and chemical damage to nucleic acids will be studied by using small oligonucleotides of defined sequence (337) which can be hybridized to mutant single strands for transfection assays to test the biological effect of the damaging agent (81, 89, 172, 240). More restriction enzymes will be isolated to be able to isolate smaller and smaller specific fragments of DNA.…”

Initiation Specificity of the Poly(cytidylic acid)‐Dependent Qβ Replicase Activity

“…The template specificity has been well characterized with the natural RNAs such as QP RNA, RNA complementary to Qp RNA, QB RNA 'variants' and a 6-S RNA found in QP-infected Escherichia coli cells [2]. The molecular basis, however, for the highly specific interaction of QP replicase with these natural templates is unknown even though the subunit structure of the enzyme [3,4] as well as the nucleotide sequence of the relevant parts of Q j RNA have been analysed [5]. The prerequisites for synthetic ribopolymers to be utilized as template by the poly(C)-dependent QP replicase activity are less well defined.…”