Leeway and constraints in the forced evolution of a regulatory RNA helix.

Abstract

The start of the coat protein gene of RNA phage MS2 adopts a well‐defined hairpin structure of 12 bp (including one mismatch) in which the start codon occupies the loop position. An earlier expression study using partial MS2 cDNA clones had indicated that the stability of this hairpin is important for gene expression. For every ‐1.4 kcal/mol increase in stability a 10‐fold reduction in coat protein was obtained. Destabilizations beyond the wild‐type value did not affect expression. These results suggested that the hairpin was tuned in the sense that it has the highest stability still compatible with maximal ribosome loading. Employing an infectious MS2 cDNA clone, we have now tested the prediction that the delta G 0 of the coat protein initiator helix is set at a precise value. We have introduced stabilizing and destabilizing mutations into this hairpin in the intact phage and monitored their evolution to viable species. By compensatory mutations, both types of mutants quickly revert along various pathways to wild‐type stability, but not to wild‐type sequence. As a rule the second‐site mutations do not change the encoded amino acids or the Shine‐Dalgarno sequence. The return of too strong hairpins to wild‐type stability can be understood from the need to produce adequate supplies of coat protein. The return of unstable hairpins to wild‐type stability is not self‐evident and is presently not understood. The revertants provide an evolutionary landscape of slightly suboptimal phages, that were stable at least for the duration of the experiment (approximately 20 infection cycles).(ABSTRACT TRUNCATED AT 250 WORDS)

Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus.

“…The phages that are recovered from such cDNA mutants are revertants carrying destabilizing suppressor mutations that increase CP yield. Eventually, the revertants reach wild-type titer by additional substitutions that further adjust the stability of the helix to the wild-type value (8).…”

“…Subsequent infection cycles start with the inoculation of about 107 GM-I cells with 106-108 pfu. Phages present in 1 ml of supernatant were precipitated with PEG and the RNA in the region of interest was sequenced by primer extension as described before (8). The sequence of revertant 2.4 was also verified by double-stranded DNA sequence analysis of two independent reverse transcription-PCR fragments.…”

“…In a previous evolutionary experiment we could show that the thermodynamic stability of this hairpin is of great importance to the fitness of the phage. Changes from its wild-type AG' value immediately triggered the selection of suppressor mutations in which the original stability, but not the original sequence, was restored (8). The maturation-gene terminator-helix (Fig.…”

Stem-loop structure in the 5′ region of potato virus X genome required for plus-strand RNA accumulation

“…In contrast, shortening of the SC region (mutant SC9) by four base-pairs decreases RNA accumulation, but to a lesser extent. Similarly, Olsthoorn et al (1994) observed that increased hairpin stability was more detrimental than reduced stability for bacteriophage MS2 gene expression. They also reported that MS2 with mutations in a hairpin containing the start codon of the CP gene reverted to a wildtype stability but not to wild-type sequence.…”

Four RNA families with functional transient structures

“…8 They noticed that the structural features are generally preserved, albeit some variations in the W arm comparing the two groups and bulge shifting in the S arm comparing MS2 and M12. 8,58,59 They also reported covariation being observed in all arms and, most importantly, in the LDI; particularly, the amount of covariations present in Group I and II are similar. 8 In addition, they were able to employ the secondary-structure prediction program MFOLD (GCG sofware, Genetics Computer Group, Madison, WI) to predict the cloverleaf structure.…”