Structure-function studies of lambda and Phage22 N antiterminators

Date of Completion

January 2000


Biology, Microbiology




The mechanism by which antiterminators transform RNAP to the termination-resistant form remains an enigma to this day. As a first step towards understanding the mechanism of antitermination, a structure-function analysis of two well-known antiterminators lambda N and Phage22 N, from the lambdoid phage family was carried out, using a genetic approach. N proteins can be broadly divided into three domains—an amino terminal domain, a central domain and a carboxy terminal domain. Mutations were made in the previously identified putative RNAP interaction domain (central domain) of lambda N protein to identify residues critical in the N-RNAP interaction. Since the longterm goal in the lab has been to carry out Cys-mediated N-RNAP crosslinking, substitution mutagenesis of Cys93 was done to look for a possible replacement that would provide us with a cleaner crosslinking data. Contrary to past reports in literature, the cysteine at position 93 in λN was found to be dispensable for N function in vivo. Several positions within the central domain can tolerate cysteine substitution (Cys used to facilitate future UV-photocrosslinking experiments). A multiple mutant of lambda N that substitutes residues K43 and K45 and E49 with Ala renders the N protein partially defective. ^ Therefore most likely the N-RNAP interaction is a multi-point, multi-domain interaction. The Phage22 N protein is very different from its lambda counterpart. To test whether such disparate N proteins interact with RNAP in similar manner, a structure-function analysis of P22N was performed. A deletion analysis of P22N revealed that the P22N is a composite of two such antitermination-proficient domains. Like lambda N, antitermination. by P22N amino terminus requires presence of its cognate nut22 site. Unlike lambda N, P22N is not affected by single nus mutations. Moreover the nut22 site, possesses an N-independent antitermination function. A two-hybrid assay to identify the interaction domain of P22N in RNAP showed that P22N (like λN) binds to conserved domains F, G and H in the beta subunit of RNAP. Therefore it appears that even seemingly divergent protein sequences can interact with the same region in their target protein. ^