Figure 3. E. coli ribosomes add extra lysines on messages containing two sequential AAA, but not AAG, lysine codons.
(A) Illustration of the ribosome on the entire MKA2-Stop message. (B) eTLCs showing the peptide products resulting from translation of indicated messages with Lys-tRNAlys (but no other tRNAs or release factors) present. (C) eTLC displaying the peptide products resulting from the translation of indicated messages in the presence of Lys-tRNALys alone, or in the presence of Lys-tRNALys + factors (either RF1 or Phe-tRNAPhe) necessary for messages to be fully translated.
Figure 3—figure supplement 1. E. coli ribosomes add extra lysines to peptides translated on messages containing sequential AAA-AAG lysine codons.
TLC showing all of the peptide products resulting from translation of MKA2V-Stop, MKA3-Stop, MKA4-STOP, and MKAKGF-Stop messages with Lys-tRNAlys (but no other tRNAs or release factors) present.
Figure 3—figure supplement 2. Quantification of the percentage of translated peptide containing more lysine residues than expected.
Translation reactions were run in the presence of either Lys-tRNAlys only, or Lys-tRNAlys and other factors (Phe-tRNAPhe or RF1). All errors bars represent the standard error from at least three independent experiments.
Figure 3—figure supplement 3. T7 transcribed messages visualized on 15% denaturing PAGE gel.
(A) In vitro transcribed mRNAs used in our in vitro studies run as distinct, single bands on high-resolution denaturing PAGE gel. The RNA is visualized with methylene blue stain. (B) The mRNAs encoding consecutive AAA codons result in discrete length toeprint signatures, yielding specific bands corresponding to the full-length message on our toeprints. We also performed RACE experiments on in vitro T7 transcribed mCherry reporter mRNAs containing A18-36 sequences and found that with high frequency, our RNAs contained the expected number of As. Importantly, in both the cell-free system and in vivo, T7 RNA polymerase is responsible for transcribing the mRNAs relevant to the output. Together, these data provide strong evidence that the mRNAs utilized throughout this study are accurately transcribed by T7 RNA polymerase.