The major goal of this study is to identify and characterize new E. coli transcription antitermination systems. Antitermination is an important pos - initiation mechanism that modulates the response of RNA polymerase to pause sites and terminators. The numbers and types of antitermination systems discovered has rapidly increased in recent years. The classic systems are those involved in lambdoid phage antitermination and attenuation in biosynthetic operons. More recently, varied antitermination mechanisms hav been described for at least six other operons. These new systems make it clear that antitermination is an important mechanism for altering RNA transcription and is more commonly used in controlling gene expression than was appreciated several years ago. Another important point made by these n w antitermination systems is that there is an unexpected variety of different antitermination mechanisms encountered, and most are not yet understood in any detail. Recently, reports of antitermination in eukaryotic gene contro have also increased. One particularly interesting study involving the AIDS producing HIV-1 virus showed that the expression of HIV-1 replication genes depends upon a trans-acting protein, Tat, that is required for read through of a terminator in the leader region of the viral long terminal repeat. Thus, expanding our basic understanding of different types of antiterminati n systems and their mechanism(s) will undoubtedly lead to a better understanding of diverse controls on gene expression in many different cell and viruses. In this application, we propose to continue to isolate and characterize new transcription antiterminators from E. coli. We have developed an antitermination test plasmid system and have used it for the isolation of these antiterminators. Our initial characterizations will continue to be t e following: 1) Quick restriction mapping to size the insert and make sure t e terminator is intact. 2) Total sequence determination of small inserts by rapid plasmid sequencing. 3) Phenotypic testing to compare relative levels of terminator read through. The next level of analysis is: 1) Subclone th insert's activity to the smallest possible sequence. 2) Analyze the sequen e for any notable or recognizable features. 3) Reclone the active fragment with another terminator to test the generality of its effect. 4) Remove th promoter and the terminator to test if the active fragment has promoter activity, or alters expression from the promoter. 5) Quantitate the read through activity by assaying enzyme activity and/or message from the report r gene downstream of the terminator. We also plan to prove the mechanism of antitermination used by these new antiterminators by performing a thorough mutational analysis of selected antiterminators, isolating suppressors of t e manufactured mutants to identify cellular factors which interact with the antiterminator, mapping the antiterminators and isolated suppressors, determining the interactions between the antiterminator and terminator, examining the role of translation and the transcribed RNA in antiterminatio , and by testing specific models for the mode of action of several different types of antiterminators we have isolated.
| Squires, C; Squires, C L (1992) The Clp proteins: proteolysis regulators or molecular chaperones? J Bacteriol 174:1081-5 |
