Translation Reinitiation Research Articles

Regulated in Vitro Synthesis of Escherichia coli Tryptophan Operon Messenger Ribonucleic Acid and Enzymes

DNA-dependent synthesis of tryptophan (trp) operon mRNA and enzymes was studied in vitro. With φ80 trpEDCBA template DNA trp operon transcription was initiated primarily at a phage promoter estimated to be approximately 7400 to 8000 nucleotides from trpE. Sequential transcription of trp mRNA in the correct direction proceeded at a rate of about 19 nucleotides per s. Trp mRNA resulting from read-through transcription from the phage promoter was larger than 23 S. Coupled sequential synthesis of trp operon enzymes occurred at a rate of approximately 4 amino acids per s. Experiments were conducted to determine if ribosomes discharge from polycistronic trp mRNA following translation termination at natural chain termination codons. Kasugamycin was used to inhibit reinitiation of translation. Kasugamycin shut down of steady state translation required 6.5 min for trpE enzyme, 7 min for trpD enzyme, and 3 min for trpA enzyme. These results indicate that ribosomes which translate trpE mRNA must discharge and reattach in order to translate trpD mRNA, and that ribosomes that translate trpD mRNA must discharge and reattach in order to translate trpA mRNA. Kasugamycin inhibition of trpD enzyme and trpA enzyme synthesis under appropriate conditions of sequential transcription and coupled translation provided further evidence for obligatory discharge and reattachment of ribosomes. Enzyme synthesis coupled to trp operon-promoted transcription from λh80 trp template DNA was repressible by tryptophan in reaction mixtures containing S-30 extract from trpR+ cells. In contrast, enzyme synthesis coupled to read-through transcription from φ80 trp template DNA was essentially irrepressible. It is concluded that read-through transcription is not repressible in vitro. Trp operon mRNA transcribed from a λh80 trpEDCBA DNA template was smaller than 23 S. The polar mutation trpC6 was expressed in vivo in phage λh80 trpEDCBA 190-9 but not in phage φ80 trpEDCBA 190. Read-through transcription of the trp operon occurs in the latter phage but not in the former. Polarity was not expressed in vitro using template DNA from either phage. Trp operon mRNA was extremely stable in vitro; the half-life varied between 30 min and more than 2 hours.

Protein Hal: partial deletion of a " " immunoglobulin gene(s) and apparent reinitiation at an internal AUG codon.

Protein Hal is a human gamma4 heavy-chain disease protein whose molecular weight is reduced from 55,000 to 25,000 in 6 M guanidine due to the lack of disulfide bonds between heavy chains. Studies of aminoacid sequence indicate that it contains a gap of about 240 residues, starting 10 residues from the N-terminal end and including the rest of the Fd fragment, as well as the hinge region. Normal sequence apparently resumes at a methionine residue (position 252) in the second constant domain (C(H)2) and seems normal from there to the carboxyl end of the molecule. These results imply that reinitiation of translation at an internal AUG codon occurs in protein Hal.

Translational restarts: AUG reinitiation of a lac repressor fragment.

An early, spontaneous amber mutation in the lac i-gene allows translational reinitiation, which results in a mutant lac repressor. Comparison of the amino-terminal sequence of this mutant repressor with the partial amino-acid sequence of the wild-type lac repressor shows that reinitiation occurs at the first internal AUG codon, and results in a mutant protein lacking 42 residues at the amino-terminal end. This protein binds the inducer isopropyl-beta-D-thiogalactoside with normal affinity, and is capable of maintaining a tetrameric structure; however, it does not repress in vivo. These data suggest that the amino-terminal portion of the wild-type lac repressor is necessary either for direct binding to the lac operator or for the correct conformation for binding to DNA.