A functional hybrid ribosome binding site in tryptophan operon messenger RNA of Escherichia coli

Christie, Gail E.; Platt, Terry

doi:10.1016/0022-2836(80)90195-3

Cited by 12 publications

(7 citation statements)

References 18 publications

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“…pAROP is a derivative of pBR322 in which a Bgl II linker has been inserted into the Pvu II site, disrupting the repressor of the primer gene (13). The tryptophan (trp) promoter-operator and Shine-Dalgarno sequences, derived from pLD102 (14), were inserted into the Cla I site of pBR322, generating the expression vector pKGP36.trp. A derivative vector ofthe M13 phage, mp2348, was provided by W. Barnes (15).…”

Section: Methodsmentioning

confidence: 99%

Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.

Ivanoff¹,

Towatari²,

Ray³

et al. 1986

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

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We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotidedirected mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histide-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.Poliovirus, a small RNA virus, is a member of the picornavirus family, which includes rhinovirus, encephalomyocarditis virus, and foot and mouth disease virus. During the viral life cycle, the genomic RNA is initially translated into a >200-kDa polyprotein, which, while nascent, is cleaved to a series of polypeptide intermediates, termed P1, P2, and P3. Further processing yields the majority of mature viral products (structural and nonstructural) and is dependent on a viral protease(s). The poliovirus P3 intermediate consists of three nonstructural proteins-the genome-linked virion protein (VPg), the protease (3C), and the replicase (3D)-and two proteins of unknown function (3C' and 3D') (1, 2). Hanecak et al. (3) have demonstrated that the 3C region of the poliovirus genome represents the core sequence responsible for the proteolytic processing (at Gln-Gly) of the precursor polypeptides. In addition, the poliovirus 3C protease has been reported to be autocatalytic (4), in agreement with results for encephalomyocarditis virus (5) and foot and mouth disease virus (6).The enzymatic mechanism of these viral proteases has not been well characterized. Inhibitors such as iodoacetamide, N-ethyl maleimide, and para-chloromercuribenzoate have implicated a cysteine residue in the active site (7,8). However, diisopropylphosphofluoridate, an inhibitor of serine proteases, has also been reported as an effective inhibitor of poliovirus protein processing (9). A comparative study (10) of the derived protease proteins from several picornaviruses as well as the related plant virus cowpea mosaic virus (11) revealed one region of significant homology located in the carboxyl third ofthe protein. Within this region a cysteine and a histidine residue have been strictly conserved. These observations suggest that the picornavirus proteases most probably belong to the class of cysteine proteases.To obtain sufficient quantities of the protease proteins for study, we have engineered a cDNA clone of the poliovirus 3C-3D region into an Escherichia coli expression vector, which produced the protease polypeptides in high yield...

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Section: Methodsmentioning

confidence: 99%

Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.

Ivanoff¹,

Towatari²,

Ray³

et al. 1986

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

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“…The plasmid pWU11, containing the trp terminator (trp t) next to the trp promoter, is described in Wu et al (14). The plasmid pLD102, which was used in construction of synthetic terminators, is described in Christie and Platt (15 followed by four T residues (Fig. la).…”

Section: Methodsmentioning

confidence: 99%

Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro.

Christie¹,

Farnham²,

Platt³

1981

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

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Termination of transcription by Escherichia coli RNA polymerase in vitro appears to depend primarily on two structural features of the termination site-a G+C-rich region of dyad symmetry and a series of terminal uridine residues in the transcript. To determine whether these two features are sufficient to specify p-independent termination in vitro, we have introduced new sequences within a tryptophan (trp) operon structural gene to create two sites with these characteristics. Transcription with wild-type RNA polymerase in vitro demonstrates that discrete termination occurs at one of these new sites, although at a low level. Use of the mutant RNA polymerase rpo203, which is more sensitive to certain weak terminators than is the wild-type enzyme, increases termination at both sites. We have compared the activity ofour synthetic terminators with those ofseveral termination sites in the E. coli trp operon. Under normal conditions of transcription in vitro, termination becomes more efficient with an increase in the length of the stem in the RNA hairpin or an increase in the number of consecutive uridine residues. Transcription with the rpo203 polymerase and with ribonucleotide analogs gives changes consistent with these general trends. These results support a model for termination involving separate but essential roles for the RNA hairpin and the stretch of uridines in the transcript.Termination of transcription by Escherichia coli RNA polymerase generally occurs at specific sites on a DNA template. Although termination is modulated by the transcription termination factor p in vivo (1), many terminators function in vitro in the absence of p. Sequence comparison of p-independent termination sites reveals common features (2, 3). These include a G+C-rich region of dyad symmetry, which can result in an RNA hairpin, and a series of terminal uridine residues in the transcript immediately 3' to the region of dyad symmetry. Our studies have suggested that these two features reflect the involvement of both RNA-RNA and RNA-DNA interactions in termination at p-independent sites (4). The presumed role of the RNA hairpin in inducing a pause by RNA polymerase is supported by experimental evidence (2,5,6). The extreme instability ofrU-dA base-pairing (7) probably enhances the release ofthe nascent transcript at the run ofuridines. According to our model, the balance between elongation and dissociation ofRNA polymerase at a termination site depends on the potential stability of the RNA hairpin and the weakness of the interaction between the terminal region ofthe transcript and the template.Results obtained by varying conditions of transcription in vitro are consistent with this model. Termination can be affected not only by nucleotide sequence, but also by the nature of the incorporated ribonucleotides and the RNA polymerase (4, 8-10). Our previous studies have focused on the trp attenuator termination site (trp a) as a model system. Removal offour of the eight terminal uridine residues from this site by the deletion trp a14...

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“…The region of the synthetic IFN gene fragment 5' to the translation initiation codon was removed because (a) it contains a BamHI cohesive end, unsuitable for joining to the ClaI site of the trp promoter and (b) the nucleotide sequence around the BamRT site and before the translation initiation codon is unlike that in any natural translation initiation region described to date. This editing of the IFN gene was achieved by cutting at a Sau3A site in the codons for amino acids 3 and 4 of the IFN-a1 polypeptide and the use of synthetic oligonucleotides to reconstruct the beginning of the IFN gene and the natural high efficiency translation initiation region of the t4C gene in the trpID102 deletion mutant (34). 51 -AAGCGUAUCGACGAUG.UGU.GAU.…”

Section: Resultsmentioning

confidence: 99%

“…51 -AAGCGUAUCGACGAUG.UGU.GAU. in which the overlined region is equivalent to the first SD sequence of the trp operon, the underlined sequence is the first (Met) codon of the synthetic IFN gene and the intervening sequence is identical to that in the first translation initiation region of the trILD102 mutant t operon (34).…”

Section: Resultsmentioning

confidence: 99%

“…These partially complementary oligonucleotides were designed to join ClaI and BglII cohesive ends, to regenerate the coding sequence for the first two amino acids of the synthetic IFN-a1 gene (Met and Cys) and to give a sequence between the trp promoter SD sequence and the IFN Met translation initiation codon which is identical to that found in the trLD102 deletion mutant hybrid ribosome binding site (34). (This translation initiation region is known to be highly effective in vivo (16).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The construction of a syntheticEscherichia coli trppromoter and its use in the expression of a synthetic interferon gene

Windass

Newton²,

Maeyer‐Guignard

et al. 1982

Nucl Acids Res

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A functional hybrid ribosome binding site in tryptophan operon messenger RNA of Escherichia coli

Cited by 12 publications

References 18 publications

Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.

Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.

Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro.

The construction of a syntheticEscherichia coli trppromoter and its use in the expression of a synthetic interferon gene

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