0000000000122245
AUTHOR
Normunds Licis
RNA In Vitro Synthesis by Phage T7 DNA-Dependent RNA Polymerase
The purpose of this chapter is to introduce beginners in molecular biology to RNA transcription by phage T7 DNA-dependent RNA polymerase. The work outlined here includes the transcription procedure of plasmid vectors or PCR-amplified DNA templates, the purification and identification of RNA products by sequencing with reverse transcriptase.
Forced Retroevolution of an RNA Bacteriophage
AbstractThe operator hairpin ahead of the replicase gene in RNA bacteriophage MS2 contains overlapping signals for binding the coat protein and ribosomes. Coat protein binding inhibits further translation of the gene and forms the first step in capsid formation. The hairpin sequence was partially randomized to assess the importance of this structure element for the bacteriophage and to monitor alternative solutions that would evolve on the passaging of mutant phages. The evolutionary reconstruction of the operator failed in the majority of mutants. Instead, a poor imitation developed containing only some of the recognition signals for the coat protein. Three mutants were of particular inter…
Long-range translational coupling in single-stranded RNA bacteriophages: an evolutionary analysis
In coliphage MS2 RNA a long-distance interaction (LDI) between an internal segment of the upstream coat gene and the start region of the replicase gene prevents initiation of replicase synthesis in the absence of coat gene translation. Elongating ribosomes break up the repressor LDI and thus activate the hidden initiation site. Expression studies on partial MS2 cDNA clones identified base pairing between 1427-1433 and 1738-1744, the so-called Min Jou (MJ) interaction, as the molecular basis for the long-range coupling mechanism. Here, we examine the biological significance of this interaction for the control of replicase gene translation. The LDI was disrupted by mutations in the 3'-side an…