Studies of the Molecular Mechanism of Attenuation in the Threonine Operon of Escherichia Coli Using Site-Directed Mutagenesis (Growth Dependent Expression, Translation, Ribosome Interference, Termination, Transcriptional Pausing)

185 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1986.The threonine (trh) operon of E. coli is regulated by an attenuation mechanism. The proposed attenuation model indicates that premature transcription termination occurs at the attenuator unless a translating ribosome stalls at the regulatory threonine and isoleucine codons in the leader RNA. Oligodeoxy-ribonucleotide directed in vitro mutagenesis was used to introduce a dG in place of a dA residue at position 110 of the leader sequence of the thr operon. This mutation removes the normal translation stop codon of the putative leader peptide. The next in-frame stop codon in the sequence lies 36 nucleotides downstream at position 146 in the attenuator region. The mutation was constructed such that a thr-lacZ fusion could be cloned into (lamda)gt4 phage vector and the recombinant used to prepare single copy (lamda)lysogens in three different strains of E. coli K12--MC4100, MO and RZ201. The mutation causes overexpression of the thr operon relative to the wild type, especially in the presence of the regulatory amino acids, Thr and Ile (presumably because the ribosome directly blocks the formation of the termination structure 3:4). These data provide evidence for the direct role of translation in the control of thr operon expression and are thus in accordance with the proposed attenuation model for the regulation of this operon. The doublings per hr ((mu)) of the lysogens were varied by growing the cells at 37(DEGREES), 30(DEGREES), and 24.5(DEGREES)C. The extent of the overexpression of the mutant in all three strains was inversely correlated with their (mu) values, an effect which becomes more pronounced with decreasing (mu). In contrast, the expression of the wild type thr operon was decreased only slightly with the lowering (mu) in all three strains. The results may be interpreted in terms of the involvement of transcriptional pausing and translational-transcriptional coupling in the expression of the thr operon.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD