Molecular cloning and sequence analysis of trp-lac fusion deletions.

DNA fragments containing deletions that fuse the trp operon to the lac operon were cloned and the end-points of the fusions were determined. The results from DNA sequence analysis correlated well with those from genetic, biochemical and physiological studies previously reported. The sequence data from this study, in combination with the known properties of these fusion strains, provided information on: (1) the precise lac operon distal boundary of the lac operator; (2) the nature of the trp operon terminator; and (3) the messenger RNA sequences that result in inhibition of lacZ translation initiation in trp-lac fused mRNAs.

lacZ translation initiation mutations.

Sixteen single point mutations near the beginning of the lacZ gene have been isolated and their effect on lacZ expression has been measured. Five mutations were obtained that alter a potential stem-and-loop structure in the messenger RNA that masks the initiation codons. Formation of this stem-and-loop is a result of transcription of DNA sequences introduced during the cloning of the lac regulatory region. The mutations isolated were then moved into a background that deleted this structure. Analysis of these mutations indicated that the secondary structure inhibited lacZ expression 5.8-fold and that either single point mutations or a 9 base-pair deletion could relieve this inhibition completely. In addition, it was found that an A to C transversion in the first base following the initiation codon (in the absence of the inhibitory secondary structure) decreases lacZ expression almost twofold, whereas C to U transitions in the next two positions have negligible effects. Mutations were also obtained that either increase or decrease the length of the Shine-Dalgarno sequence. The effects of these mutations were studied in the presence or absence of the secondary structure that involves the two initiation codons. It was found that when translation initiation was inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence increased lacZ expression 2.8-fold and decreasing the length of this sequence reduced lacZ expression 12-fold. When translation initiation was not inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence had no effect and decreasing the length of this sequence only reduced lacZ expression sixfold. The mechanistic implications of these results are discussed. Two initiation codons are located in the beginning of the lacZ gene, 7 and 13 bases from the Shine-Dalgarno sequence. NH2-terminal sequence analysis indicated that the majority of the protein synthesized initiate at the first initiation codon in the wild-type lacZ gene (in agreement with results reported previously by J. L. Brown and his colleagues). Upon introduction of sequences that result in a change in the mRNA secondary structure, both initiation codons are used in almost equal amounts. Three mutations and two pseudorevertants were obtained, which are located in the first initiation codon. It was found that when the first initiation codon is changed from AUG to GUG, translation initiation is decreased tenfold at that codon.(ABSTRACT TRUNCATED AT 400 WORDS)

Abortive initiation and long ribonucleic acid synthesis.

In vitro transcription assays have been used to study the rate of ribonucleic acid (RNA) synthesis from the Escherichia coli lactose promotor mutant lacL8UV5 contained on a 203-bp (base pair) restriction fragment. The half-life of long (63-base) RNA production from heparin-resistant RNA polymerase-promotor complexes was found to be related to the amount of oligonucleotides released during the initiation process (abortive initiation). Studies indicate that once a ternary complex between the promoter, RNA polymerase, and a newly synthesized RNA seven and nine nucleotides long is formed, abortive initiation is reduced and the rate of synthesis of long RNAs is increased. The promoter for the left inverted repeat of the transposable element Tn5 was also examined. It was observed to have a much slower rate of production of long RNAs, and it released oligonucleotides 4 times as often as the lactose promoter. The correlation between the amount of abortive initiation and the half-time of long RNA production is discussed.

Oligonucleotide mutagenesis of the lacPUV5 promoter.

Synthetic oligonucleotides were used to introduce mutations into the lacPUV5 promoter. Four mutations were obtained at positions -13, -14, and -15, with respect to the transcriptional start site. The effects of these mutations were measured in vivo and the results are discussed with respect to the consensus sequence and other promoter mutations located in this region.