Fig 4. Mutations in the 5’-UTR of gene II mRNA in M13KE.

The normal 5’-UTR region of the mRNA is shown, containing the gene II operator sequence, the Shine-Dalgarno sequence, and the start codon. The nucleotide numbering corresponds to M13KE. Thirteen different spontaneous 5’-UTR mutations are indicated above their respective nucleotides within the 5’-UTR (black and red). The majority of these mutations were previously reported [41], and were discovered in one of five ways: (i) in Ph.D.-7 or Ph.D.-12 phage display experiments that used Zn²⁺ as a target, (ii) during serial amplification of the Ph.D.-7 library, (iii) in a 135 minute screen of amplified Ph.D.-7 or Ph.D.-12 libraries, (iv) during amplification of M13KE, or (v) as a clone that contaminated a concurrent experiment in our lab. The mutations G6793T and G6792C are new to this publication, and were discovered via method (iii). The T6797C mutation was synthetically introduced using an insert containing the mutation, and was accompanied by a concomitant deletion, T6789Δ (both green). The mutations shown in red were made using a randomized synthetic insert, but had already arisen spontaneously in our experiments. Several mutations have been discovered repeatedly with different displayed peptides (or no peptide), including the two clones reported herein, Ph-VTAHGGR and Ph-SDLVLRP, which have new peptides but previously found mutations. These repeated mutations are listed here as: Mutation (Number of times observed): G6813A (4), C6810T (2), A6809C (3), T6798C (4), T6798Δ (3), G6793A (3), G6793Δ (2), G6792T (4). Two additional mutations arose through method (i) and are not shown here because they are upstream of the mRNA sequence: G6748Δ (in the gene II promoter) and C6589T (in the lacZα insert) [41].