Deletions of distal sequence affect termination of transcription at the end of the tryptophan operon in E. coli

Wu, Anna M.; Chapman, Alger B.; Platt, Terry; Guarente, Leonard; Beckwith, Jonathan

doi:10.1016/0092-8674(80)90073-2

Cited by 43 publications

(30 citation statements)

References 13 publications

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“…Deletion of sequences close to the last bases (deletion 44-77 or deletion [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54]) has little effect on activity, whereas deletion of bases further upstream (deletion 14-35, deletion 27-62, or deletion 7-43) allowed substantial read-through. These data indicate that for both the CYCI and UTRI terminators the most critical regions lie tens of bases upstream of the last template-derived base of the mRNAs.…”

Section: Results the Sequence In The Ava Ll-fnudii Restriction Fragmentmentioning

confidence: 99%

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Mutational analysis of a yeast transcriptional terminator.

Osborne

Guarente

1989

Proc. Natl. Acad. Sci. U.S.A.

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We have isolated and mutagenized a DNA fragment from Saccharomyces cerevisiae that specifies mRNA 3' end formation for the convergently transcribed CYCI and UTRI genes. An in vivo plasmid supercoiling assay previously showed that this fragment is a transcriptional terminator, and "run-on" assays shown here are consistent with this interpretation. The poly(A) sites in the mRNAs formed by the fragment are the same whether the fragment resides at the native location or at a heterologous location. No single linker substitution abolishes the fragment's activity, whereas certain large, nonoverlapping deletions have strong, deleterious effects. Therefore, the yeast terminator behaves more like rhodependent bacterial terminators than terminators of higher eukaryotes. That a number of deletions or substitutions have different effects in the two orientations suggests that the fragment contains the sequences of two, unidirectional terminator elements.The primary structure of the eukaryotic mRNA is due to precise sites of transcriptional initiation and RNA processing events, including splicing and end formation (reviewed in refs. 1-4). The clearest models of mRNA 3' end formation emerge from genetic and biochemical experimentation in metazoan cells. Three general events have been described. (i) Transcription by RNA polymerase II appears to terminate hundreds of base pairs downstream from the eventual junction of the template-derived RNA and the poly(A) tract (5-8). DNA fragments from those downstream regions that act as terminators have been isolated, but their fine structure and their mode of action has not yet been elucidated (9). (ii) The sequence AAUAAA in the RNA directs cleavage 10-30 bases 3' to the hexanucleotide element (10-15). Extensive mutagenesis of this sequence and its immediate environment indicates that AAUAAA is essential for proper processing and that sequences immediately 3' to the cleavage site are also required (16)(17)(18)(19). (iii) The cleaved RNA serves as a substrate for processive polyadenylylation (20, 21). Macromolecular complexes that carry out the latter two reactions have been isolated, and small nuclear ribonucleoproteins are thought to participate (12,21,22). Remarkably, evidence has been provided that indicates that all of these processes are coupled in vivo, as mutations within the AATAAA strongly decrease the efficiency of distant transcriptional termination (23-25).How similar is 3' end formation in yeast to what occurs in higher cells? The consensus element TAG. .TAGT. .TTT has been proposed as a key element in termination for many genes, including CYCI (iso-1-cytochrome c) of Saccharomyces cerevisiae (26). To begin an analysis of termination in yeast, we chose to study an 83-base-pair (bp) fragment past the 3' end of CYCI that includes the consensus element. CYCI and an adjacent gene, UTRI, are convergently transcribed and 3' ends for both mRNAs fall within the 83-bp region (Fig. 1).We showed previously that transcription of CYCJ-lacZ on a plasmid in S. cerevisiae resulted in...

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Section: Results the Sequence In The Ava Ll-fnudii Restriction Fragmentmentioning

confidence: 99%

“…These terminators are pyrimidine-rich tracts, in which small substitutions or deletions have little or no effect (43)(44)(45)(46). In some of these cases, the terminated transcript is processed at a site several hundred bases upstream of the termination site.…”

Section: Discussionmentioning

confidence: 99%

Mutational analysis of a yeast transcriptional terminator.

Osborne

Guarente

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…(iii) Under controlled in vitro conditions, viral RNA 93-95 nucleotides long can be released from the template (unpublished data). (iv) Discrete aborted RNAs have been observed in Ad-2-infected cells that most likely represent premature termination products (I1).-By analogy to the attenuationmechanism in prokaryotes, it is possible that a number of additional features are involved in the process of premature termination in.SV40, such as the presence of G+ C-rich sequences distal to the termination site that can form secondary structures (29,37,38), protein that accentuates the termination event [a likely candidate is the "agnoprotein" (36)], and protein that functions as an antiterminator [a likely candidate is one of the early gene products (29)]. Moreover, the specific tertiary structure ofthe SV40 minichromosome exhibiting a "gap" in, the region encompassing the initiation and attenuation sites could also, play a role in the attenuation mechanism (44)(45)(46).…”

Section: Length Distributions Ofmentioning

confidence: 99%

Site of premature termination of late transcription of simian virus 40 DNA: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Skolnik-David¹,

Hay²,

Aloni³

1982

Proc. Natl. Acad. Sci. U.S.A.

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Sedimentation analysis of pulse-labeled RNA synthesized in nuclei isolated from simian virus 40-infected cells revealed an abundance of short cellular and viral RNAs. The relative amount ofthe short chains is increased in nuclei isolated from cells treated with 5,6-dichloro-l-.l-D-ribofuranosylbenzimidazole (DRB). The short viral RNAs were purified by hybridization to and elution from simian virus 40 DNA on filters, and their sizes were determined by gel electrophoresis. A major band of 93-to 95-nucleotide-long RNA was observed along with additional minor bands. Identical bands were revealed when the viral RNA was purified from nuclei ofcells pretreated with DRB. The major band was identified as an aborted transcript of a RNA that initiated at the major initiation site (nucleotide 243). We have found that the DNA region where the RNA stops is A+T rich and is immediately preceded by a G+C-rich region that exhibits dyad symmetry, resembling the termination signal in prokaryotes. These observations show that RNA polymerase II responds to the same termination signal as the prokaryotic enzyme and suggest that a mechanism of attenuation regulates simian virus 40 late transcription.Transcription-termination sites in prokaryotes have several common features: G+C-rich sequences preceding the stop site, from which a stem-and-loop structure can be formed, and uridine residues in the terminus of the RNA transcripts (for review, see refs. 1-7). It has been suggested that the stem-andloop structure causes retardation of polymerase movement through the termination region, whereas the uridine residues facilitate the release ofthe transcripts (8). This implies a general mechanism for transcription termination which is based in part on the physical chemistry of nucleic acid interactions. In eukaryotes, the mechanism of termination of RNA polymerase II transcription is not yet known, and the sequences that constitute terminators have not been determined.In prokaryotes, transcription termination can occur within as well as at the end of the operon. Termination within the operon causes premature termination ofthe transcripts and quantitatively regulates the level of gene expression, by selectively reducing the transcription ofdistal portions of the operon. This mechanism has been termed "attenuation" (3). It is thought that regulation is achieved by altering the frequency with which transcription crosses the termination site.It has been suggested that the attenuation mechanism also quantitatively regulates gene expression in eukaryotes (9-15). Quantitative transcriptional changes that occur during the cell cycle and cell differentiation have been attributed to this mechanism (16). Observations ofaborted RNA in whole nuclear RNA (9, 10), in adenovirus type 2 (Ad-2) (11)(12)(13), and in simian virus 40 (SV40) (14, 15) provided experimental support for the existence of the attenuation mechanism in animal cells. Moreover, these studies have shown that the adenosine analog 5,6-dichloro-l-,-D-ribofuranosylbenzimidazole (DRB) enha...

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“…In this way, we selected several small deletions, for example, RT38 in Figure 4, that removed the mRNA termination signal. In collaboration with Terry Platt's laboratory, we were able to use the small deletions to identify where in the DNA sequence termination of trp mRNA takes place, showing that it was considerably downstream from a previously proposed location (55).…”

Section: Trp-lac Fusions: Backward and Forwardmentioning

confidence: 99%

Fifty Years Fused to Lac

Beckwith

2013

Annu. Rev. Microbiol.

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I recount the history of how I became interested in the use of gene fusions for studying biological problems. Initially, selections for mutations that would restore function to an inactivated lac operon unexpectedly yielded fusions in which lac was expressed from the controlling elements of upstream genes. Subsequently, by chance, I generated strains in which the lac operon was transposed from its normal position on the chromosome to a position close to the trp operon, thus facilitating sets of useful fusions of the two operons. The development of a more generalized technique for obtaining fusions by my student Malcolm Casadaban opened up a much broader set of biological problems that could be approached with fusions. Work on these problems included the study of protein translocation across membranes, the analysis of membrane protein topology, and the discovery of the pathway of electron transfer that leads to disulfide bond formation in proteins.

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Deletions of distal sequence affect termination of transcription at the end of the tryptophan operon in E. coli

Cited by 43 publications

References 13 publications

Mutational analysis of a yeast transcriptional terminator.

Mutational analysis of a yeast transcriptional terminator.

Site of premature termination of late transcription of simian virus 40 DNA: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Fifty Years Fused to Lac

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