Frank Lee

Nucleotide sequence of the 5' end of tryptophan messenger RNA of Escherichia coli.

Abstract The 5′-terminal sequence of the tryptophan (trp) operon messenger RNA of Escherichia coli was determined by a combination of in vivo and in vitro 32P-labeling techniques. A leader sequence of approximately 166 nucleotides precedes the start codon for the polypeptide specified by the operator-proximal structural gene, trpE. The leader sequence contains an AUG codon (positions 27 to 29) in a region which binds ribosomes (Platt et al., 1976) and is followed in phase by the tandem chain termination codons, U-A-A-U-G-A (positions 120 to 125). The inferred DNA sequence has a region of high G + C content (positions 126 to 137) followed by a region of high A + T content (positions 138 to 156). Studies in vivo (Bertrand et al., 1976) and in vitro (Lee et al., 1976) suggest that transcription termination occurs in the vicinity of these G + C and A + T-rich regions and that termination may be regulated (Bertrand & Yanofsky, 1976).

Interaction of the trp repressor and RNA polymerase with the trp operon

Abstract An in vitro transcription system for the tryptophan operon of Escherichia coli was used to determine the mechanism by which trp † repressor inhibits transcription of the operon. It was found that trp repressor-binding prevents binding of RNA polymerase to the operon. It was also observed that when polymerase is prebound to the trp promoter, repressor cannot prevent it from transcribing. These results suggest the existence of a functional overlap between trp promoter and trp operator regions and indicate that repressor acts by preventing RNA polymerase attachment to the promoter region of the operon.

Termination of transcription in vitro in the escherichia coli tryptophan operon leader region

Abstract Transcription of the wild-type tryptophan (trp) operon of Escherichia coli was examined in vitro. Virtually all RNA polymerase molecules which initiate transcription at the trp promoter cease transcription within the leader region of the operon after synthesizing about 145 nucleotides of leader RNA, and thus rarely transcribe the structural genes of the operon. Transcription stops with approximately equal frequency at either of two adjacent nucleotide pairs within an A + T-rich region, giving rise to transcripts with U-rich 3′ termini. The site of transcription termination is in a segment of the leader region proposed on the basis of genetic and biochemical evidence to contain a new regulatory element, a transcription attenuator, which functions in controlling the maximum level of expression of the operon.

Comparison of the nucleotide sequences of the initial transcribed regions of the tryptophan operons of Escherichia coli and Salmonella typhimurium

The nucleotide sequences of the initial transcribed regions of the tryptophan ( trp ) operons of Escherichia coli and Salmonella typhimurium were determined. These regions include the leader segment of each operon and the segment coding for the first approximately 30 amino acid residues of the respective trpE polypeptide chains. Similar nucleotide sequences are found in the G+C and A+Trich sequence blocks in the vicinity of the transcription-termination control sites contained within each leader region. In addition, potentially translatable sequences are present in each leader region that correspond to similar 14-residue peptides, each containing tandem tryptophan residues at positions 10 and 11. The RNA transcripts synthesized from the leader regions of the two trp operons could form stable “stem and loop” secondary structures in the segment corresponding to the region preceding the transcription termination site. Comparison of the trpE coding regions shows that synonymous codon differences predominate over those resulting in amino acid changes.

The attenuator of the tryptophan operon of Escherichia coli: Heterogeneous 3′-OH termini in vivo and deletion mapping of functions

The attenuator of the tryptophan (trp) operon of Escherichia coli, located between the promoter and the first major structural gene of the operon, functions as a site of regulated transcription termination (Bertrand et al., 1975). Discrete in vivo RNA transcripts, corresponding to the initial 137 to 141 nucleotides of trp mRNA, were isolated and characterized. RNase T1 digests of these transcripts yielded a series of 3′-OH oligonucleotides with the general sequence CUn-OH (n = 4 to 8), suggesting that transcription terminates in vivo at any of five adjacent base-pairs in the A + T-rich portion of the attenuator region. These 3′-OH termini are essentially identical to the 3′-OH termini of the major trp mRNA species produced in vitro (Lee et al., 1976). Internal deletions within the trp operon, with one terminus in the immediate vicinity of the attenuator, were characterized as to their effects on (1) the production of attenuator-terminated transcripts, (2) the rho-dependence of attenuator termination, and (3) the relief of attenuator termination accompanying tryptophan starvation. We find that the nucleotide sequence essential for normal termination and its control extends no more than 11 base-pairs beyond the site of termination in vivo. Deletion of a portion of the A + T-rich sequence of the attenuator reduces the frequency of termination in vivo, and eliminates termination in this region in vitro.

RNA polymerase interaction at the promoter--operator region of the tryptophan operon of Escherichia coli and Salmonella typhimurium.

Restriction fragments from the promoter-operator region of the tryptophan operon of Escherichia coli were isolated and the location of the DNA regions which interact with RNA polymerase bound at the trp promoter was examined. RNA polymerase protects from deoxyribonuclease I digestion a region of approximately 37 base-pairs centred on the transcription initiation site. RNA polymerase also protects HincII and AluI restriction endonuclease recognition sequences located 32 to 37 and 38 to 41 base-pairs, respectively, before the transcription start-site. Although RNA polymerase in the initiation complex protects approximately the first 20 base-pairs of the transcribed portion of the operon, other studies have shown that replacement of the nucleotide sequence beyond position + 1 by a foreign sequence does not significantly affect promoter function in vivo (Bennett & Yanofsky, 1978) . Promoter function of isolated restriction fragments was evaluated by: (1) the ability to act as a template for trp messenger RNA transcription in vitro and (2) the ability of RNA polymerase to specifically protect an HpaI restriction endonuclease cleavage site present in the trp promoter-operator region and contained in each of the fragments. Fragments with termini 78 base-pairs or more before the transcription initiation site have both promoter functions. A fragment which terminates 39 base-pairs before the transcription initiation site is not efficient in either function. This places the boundary of the sequence required for trp promoter function in vitro between 39 and 78 base-pairs preceding the transcription initiation site. Findings from functional analyses with fragments from the conserved Salmonella typhimurium trp promoter-operator region (Bennett et al., 1978b) suggest that the sequences required for trp promoter function in vitro are contained within the DNA segment extending 59 basepairs before the messenger RNA start-site.

Glucocorticoid Regulation of Gene Expression: Mouse Mammary Tumor Virus as a Model System

Publisher Summary Glucocorticoids—as well as other classes of steroid hormones—appear to function via the two-step model. It is generally accepted that steroids interact with a soluble receptor protein inducing a structural alteration that increases the receptors affinity for DNA or chromatin. This so-called activated form of the steroid–receptor complex accumulates within the nucleus of the cell leading to increased—and perhaps in some cases, decreased—transcription of specific genes. This chapter describes the glucocorticoid regulation of gene expression. The classes of new messenger RNAs (mRNAs) produced in response to a given steroid are in large part cell or tissue specific and their utilization in production of new proteins leads to the characteristic hormonal response of the target cell. The primary role of the steroid is to act as an allosteric effector that unmasks a DNA-binding site on the receptor protein. The chapter briefly reviews some of the studies on the glucocorticoid regulation of gene expression and presents a detailed account of the use of mouse mammary tumor virus (MMTV) in the mechanisms by which glucocorticoids regulate gene expression.