MUTATIONAL ANALYSIS OF THE CONSERVED REGION-2 SITE OF ADENOVIRUS E1A AND ITS EFFECT ON BINDING TO THE RETINOBLASTOMA GENE-PRODUCT - USE OF THE DOUBLE-TAGGING ASSAY

We have explored the feasibility of using a ''double-tagging'' assay f or assessing which amino acids of a protein are responsible for its bi nding to another protein. We have chosen the adenovirus E1A-retinoblas toma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant ELA gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We d emonstrated that this mutant exhibited little binding to pRB by the do uble-tagging assay, We also have shown that this lack of binding is no t due to any significant decrease in the level of expression of the be ta-galactosidase-E1A fusion protein. We then created a ''library'' of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that boun d to pRB, Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A (''true positives''), A variabl e number of clones appeared to bind equally well to both pRB and E1A ( ''false positives''). The DNA sequence of 10 true positive clones yiel ded the following consensus sequence: DLTCXEX, where X = any amino aci d. The recovery of positive clones with only one of several allowed am ino acids at each position suggests that most, if not all, of the cons erved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These r esults are consistent with those obtained from other sources. These da ta suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its inter action with another protein if the complex forms within bacteria, This assay,is rapid, and up to 1 x 10(6) mutations can be screened at one time.