Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda.
The product of the lambda ral gene alleviates restriction and enhances modification by the Escherichia coli K-12 restriction and modification system. An open reading frame (orf) located between genes N and Ea10 has been assigned to the ral gene. We have cloned this orf in a plasmid where its transcription is controlled by a thermolabile lambda repressor. Inactivation of the lambda repressor caused a 1000-fold reduction in K-specific restriction of unmodified lambda phage and a 100-fold increase in modification. In minicells transformed with ral+ plasmids, derepression resulted in the appearance of a polypeptide with a lower mobility than that predicted for a protein encoded by the orf attributed to ral; in a transcription and translation system in vitro DNA from a ral+ plasmid encoded a polypeptide with the same mobility. This polypeptide was absent when the plasmid DNA carried a mutant ral gene. The nucleotide sequence of this mutant gene defined two base changes, one of which inactivates the initiation codon of the orf. The K restriction endonuclease, which is also a K-specific methylase, is encoded by three genes designated hsdR, hsdM and hsdS, although the hsdR polypeptide is not essential for the methylase activity. We show that Ral enhances modification in a host strain lacking the entire hsdR gene, and lambda phages carrying the hsdM and S genes modify their own DNA inefficiently in the absence of Ral, despite the fact that derivatives of these phages provide efficient amplification of the K-specific methylase. Our data support a model in which, as a consequence of the interaction of Ral with either the hsdM or the hsdS polypeptide, the conformation of the enzyme is changed and the efficiency of methylation of unmodified target sites is enhanced. It has been postulated that Ral counteracts Rho, but in our experiments Ral did not relieve transcriptional polarity.
D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific; D005814:Genes, Viral; D009154:Mutation; D012234:Rho Factor / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*
D012234:Rho Factor; D014764:Viral Proteins; D004262:DNA Restriction Enzymes; C029602:endodeoxyribonuclease EcoK; D015253:Deoxyribonucleases, Type I Site-Specific