AN EFFICIENT PROTOCOL FOR LINKER SCANNING MUTAGENESIS - ANALYSIS OF THE TRANSLATIONAL REGULATION OF AN ESCHERICHIA-COLI RNA-POLYMERASE SUBUNIT GENE

A protocol has been developed that is capable of saturating regions hu ndreds of basepairs in length with linker scanning mutations. The effi cacy of this method stems from the design of the linker scanning mutag enesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site fo r a different restriction endonuclease. The cleavage site for one endo nuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion w ith these endonucleases and subsequent ligation results in the replace ment of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of similar to 400 bp surrounding the start site of the gene for t he beta subunit (rpoB) of Escherichia coli RNA polymerase. The transla tion of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a conside rable distance both upstream and downstream of the start site are requ ired for normal translation. Also a site that appears to be involved i n translational repression by excess beta' has been identified.