Translational reinitiation in the presence and absence of a Shine and Dalgarno sequence

Spanjaard, Remco A.; Duin, Jan van

doi:10.1093/nar/17.14.5501

Cited by 65 publications

(58 citation statements)

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“…We added to this a synthetic DNA fragment containing directional cloning sites followed by a stop͞start sequence and the coding region of kanamycin phosphotransferase (KanPT). Although coding sequences of eubacterial polycistronic messages are usually preceded by their own Shine-Dalgarno ribosomebinding sites, translation can reinitiate without a ribosomebinding site if an AUG codon is within a few nucleotides of the stop codon of a preceding translated sequence (12).…”

Section: Resultsmentioning

confidence: 99%

“…The pORF vectors contain transcription and translation start sites, followed by a cDNA cloning site and an out-of-frame ␤-galactosidase coding sequence (5,6). Recently, vectors have been constructed that translate cloned sequences in all three reading frames (pFlag⅐shift 12 , IBI), so that correct proteins are produced somewhere in the library. However, these vectors do not reduce the background of incorrect translation products, do not exclude 5Ј and 3Ј nontranslated sequences, and do not permit the selective production or purification of the correct protein sequences.…”

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confidence: 99%

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Generation of cDNA expression libraries enriched for in-frame sequences

Davis

Benzer

1997

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

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Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNAencoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.Bacterial cDNA expression libraries are made to reproduce protein sequences from a source tissue and are typically screened for antibody-binding or functional domains. Our goal is to use pooled cDNA-encoded proteins as complex antigens for making panels of monoclonal antibodies, then to isolate the epitope-encoding cDNAs. It is of great importance in such screens that the expressed polypeptides be authentic, lest the isolated clones be useless. This requires cDNA expression libraries that produce mostly authentic proteins. But translation of library cDNAs is initiated by prokaryotic signals in the vector, so cloning cDNAs in an expression vector without respect to orientation or knowledge of the correct frame will yield a majority of clones in either the wrong orientation (1͞2) or the wrong frame (2͞3), or containing 5Ј or 3Ј untranslated sequences (Ϸ1͞2). Only Ϸ8% of clones can be expected to produce authentic protein sequences.Methods have been devised that partially circumvent these problems. Directional cloning of oligo(dT)-primed cDNA fragments ensures that only forward reading frames are expressed in the library (1-4). The pORF vectors contain transcription and translation start sites, followed by a cDNA cloning site and an out-of-frame ␤-galactosidase coding sequence (5, 6). Recently, vectors have been constructed that translate cloned sequences in all three reading frames (pFlag⅐shift 12 , IBI), so that correct proteins are produced somewhere in the library. However, these vectors do not reduce the background of incorrect translation products, do not exclude 5Ј and 3Ј nontranslated sequences, and do not permit the selective production or purification of the correct protein sequences.We have designed and tested a new vector, pKE-1 (for Kosher Expression-1; pronounced picky one). By its design, cDNAs cloned in ORFs should confer kanamycin resistance upon the host, whereas...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Generation of cDNA expression libraries enriched for in-frame sequences

Davis

Benzer

1997

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

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“…This type of model has been proposed to explain the translational coupling of the rpmI and rplT genes (28). (43,44). If the two-ribosome model is valid, the effect of the premature stop codons on E expression should be related to the position of the stop codon within the secondary structure.…”

Section: Resultsmentioning

confidence: 99%

Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli

Dallmann

Dunn

1994

J Bacteriol

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Escherichia coli expresses the and E subunits of F1F0-ATP synthase at relative levels consistent with the 3:1 (,B/£) stoichiometry in the holoenzyme. The mechanism of translational control of expression of the uncC gene (£ subunit) relative to the immediately 5' uncD gene (,1 subunit) was examined. Previous expression studies and a computer analysis suggested the presence of an RNA secondary structure including the 3' end of uncD, the uncDC intergenic region, and the uncC Shine-Dalgarno sequence (S. D. Dunn and H. G. Dallmann, J. Bacteriol. 172:2782Bacteriol. 172: -2784Bacteriol. 172: , 1990 expressed from the same transcript, control of their relative levels of expression must be exerted at the posttranscriptional level. Translational control of protein biosynthesis is a method widely used for regulation of gene expression in bacteria (for recent reviews, see references 7, 20, and 33) and, in general, is exerted at the level of translation initiation rather than elongation (21, 23). For genes located in polycistronic operons, features of the translation initiation regions (TIRs) including the intrinsic sequence of the region as well as potential for mRNA secondary-structure formation largely govern the efficiency of translation (10,20,41,45).The potential for mRNA to form secondary structures provides a powerful method of control of gene expression for genes contained in polycistronic messages. Secondary structures which can sequester critical regions of a TIR limit the accessibility of the translation machinery to an mRNA and reduce expression (7,42 We have previously shown that an inhibitory effect of upstream uncD sequences on the expression of the uncC gene was correlated with a predicted mRNA secondary structure involving the uncC TIR and 72 bases at the 3' end of the uncD coding region (12). In the present paper, we report the physical determination of the secondary structure of in vitro-transcribed uncDC RNA and demonstrate that -subunit synthesis is partially coupled to translation of the uncD gene. MATERIALS AND METHODSBacterial strains and growth. The E. coli strains used in this work are listed in Table 1. Strain JM103 was used in the construction of all plasmids and was grown in Luria-Bertani medium at 37°C with shaking.Strain AN1460 was the source of purified Fj-ATPase used as a standard and was grown as described elsewhere (13). Strains AN724 and AN1008 were used in 13 and -expression studies and were grown in 2 x YT medium (31) at 37°C with shaking. When required, these cultures were supplemented with arginine, uracil, and 2,3-dihydroxybenzoic acid each to 40 ,ug/ml.

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“…S3), indicating that the SD-like sequence is required for the translational reinitiation. SD sequences are known to be required for translational coupling in E. coli (41). The overlapping region probably forms secondary structures to which free ribosomes are inaccessible.…”

Section: Discussionmentioning

confidence: 99%

Additional pathway to translate the downstream ndhK cistron in partially overlapping ndhC-ndhK mRNAs in chloroplasts

Yukawa

Sugiura

2013

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

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The chloroplast NAD(P)H dehydrogenase ( NDH ) C ( ndhC ) and ndhK genes partially overlap and are cotranscribed in many plants. We previously reported that the tobacco ndhC/K genes are translationally coupled but produce NdhC and NdhK, subunits of the NDH complex, in similar amounts. Generally, translation of the downstream cistron in overlapping mRNAs is very low. Hence, these findings suggested that the ndhK cistron is translated not only from the ndhC 5′UTR but also by an additional pathway. Using an in vitro translation system from tobacco chloroplasts, we report here that free ribosomes enter, with formylmethionyl-tRNA fMet , at an internal AUG start codon that is located in frame in the middle of the upstream ndhC cistron, translate the 3′ half of the ndhC cistron, reach the ndhK start codon, and that, at that point, some ribosomes resume ndhK translation. We detected a peptide corresponding to a 57-amino-acid product encoded by the sequence from the internal AUG to the ndhC stop codon. We propose a model in which the internal initiation site AUG is not designed for synthesizing a functional isoform but for delivering additional ribosomes to the ndhK cistron to produce NdhK in the amount required for the assembly of the NDH complex. This pathway is a unique type of translation to produce protein in the needed amount with the cost of peptide synthesis.

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Translational reinitiation in the presence and absence of a Shine and Dalgarno sequence

Cited by 65 publications

References 31 publications

Generation of cDNA expression libraries enriched for in-frame sequences

Generation of cDNA expression libraries enriched for in-frame sequences

Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli

Additional pathway to translate the downstream ndhK cistron in partially overlapping ndhC-ndhK mRNAs in chloroplasts

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