Figure 2
From: RNA mutagenesis yields highly diverse mRNA libraries for in vitroprotein evolution
Coupling Qβ replicase mutagenesis to ribosome display. The UTR, target gene, and CL region are cloned in the reverse orientation (frame 2) relative to the T7 promoter and the RQ 135-1(-) sequence to avoid intrinsic stop codons found in the modified RQ 135-1(-) sequence. Stop codons are found in frames 1 and 3 of the 5' segment and in all 3 frames of the 3' segment of the RQ 135-1(-) sequence (indicated by grey arrows and the script F1 indicating frame 1 etc.). The absence of stop codons downstream from the translational start signal is an essential requirement of ribosome display. The pathway is as follows; the SmaI digested plasmid is used as a template for transcribing mRNA via the T7 promoter sequence. Although the UTR-target gene-CL message is in the reverse orientation and not suitable for translation, the RQ 135-1(-) sequence is in the correct orientation for efficient recognition by Qβ replicase. The resulting mRNA becomes the template for Qβ replicase. Amplification of this mRNA template with Qβ replicase generates both (-) and (+) mRNA. The mRNA in the correct orientation for translation is coupled to ribosome display to produce a protein complex (target gene + CL + RQ 135-5') that remains tethered to the ribosome due to the absence of a stop codon.