Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Shinedling, Sidney T.; Gayle, Margit; Pribnow, David; Gold, Larry

doi:10.1007/bf00331582

Cited by 49 publications

(29 citation statements)

References 40 publications

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“…In such a structure the putative -35 region would be concealed within the stem (as would the extreme 5'-end of the ribosome-binding site), while the -10 sequence would be situated within the loop. Stem loops 5' to the ribosome-binding site and start codon have been described in E. coli, and implicated in mRNA stability by protecting the translation initiation complex [44]. A second region of dyad symmetry was observed 75 nucleotides 3' to the UAA termination codon, and resembles a pindependent transcriptional terminator since it is followed by a stretch of dT (five out of eight).…”

Section: Features Of Noncoding Regionmentioning

confidence: 99%

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene

Thompson

Brehm

Oultram

et al. 1990

European Journal of Biochemistry

171

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A 26-mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the Nterminus of the Closlridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2-kb PvuII -TuqI fragment and the remaining regions of the light chain and heavy chain as a 2.4-kb Sea1 -TuqI fragment and a 3.4-kb HpaI-f'~vuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N-termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.Toxigenic strains of Clostridium botulinum, an anaerobic spore-forming bacteria, produce one or more of seven immunologically distinct neurotoxins. They act primarily at the neuromuscular junction, causing paralysis by inhibiting the release of the neurotransmitter acetylcholine [I]. The botulinum neurotoxins are synthesised as a single polypeptide chain but in their most active form comprise of two asymmetric polypeptide units; a light chain (50-59 kDa) and a heavy chain (85 -105 kDa), linked by at least one disulphide bridge [2].Although botulinum neurotoxins exhibit high degrees of similarity (when compared to each other and related tetanus and diphtheria toxins) in their molecular masses and modes of action, they still remain poorly characterised at the molecular level. Proposals for the mechanism of toxic action implicates three separate functional domains. The light chain has the pharmacological activity, while the N-terminal and C-terminal of the heavy chain mediate channel formation and toxin binding, respectively. These heavy-chain, toxin-binding functional domains have been demonstrated with diphtheria toxin [3 -51 and with tetanus toxin [6 -81. Channel forming activities have been demonstrated for the N-terminus of the heavy chain with C. botulinum type A [9, 101 and there is circumstantial Correspondence to N.

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Section: Features Of Noncoding Regionmentioning

confidence: 99%

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene

Thompson

Brehm

Oultram

et al. 1990

European Journal of Biochemistry

171

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“…We have shown the latter sequence, as it lengthens the base pairing and places the conserved UUCG in a 4-base loop. Hairpin constructs 14-16 have been described (18,19). term, Terminal; int, internal.…”

Section: Cttcgg Predictsmentioning

confidence: 99%

CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.

Tuerk

Gauss

Thermes

et al. 1988

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

334

262

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The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin. Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences. In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop. Avian myeloblastosis virus reverse transcriptase, which can traverse hairpins of larger calculated stability, terminates efficiently at these CUUCGG hairpins.Thermal denaturation studies of model hairpins show that the loop sequence UUCG dramatically stabilizes RNA hairpins when compared to a control sequence. These data, when combined with previously described parameters of helix stability, suggest that T4 has utilized this loop sequence to optimize the stability of intercistronic hairpins. Fig. 1. Oligonucleotide primers were synthesized, purified, labeled at the 5' end with 32P, and used in reactions with avian myeloblastosis virus (AMV) reverse transcriptase as described (6) or in RNA blot analysis of Ml RNA as described by Maniatis et al. (7).Computer Searches. Most of the T4 sequences were in GenBank release 42.0. The sequences for gene 39, pseT, and the sequence including gene 52 and the end of rIIB (SaA9) have been published (8,9,36). All sequence manipulations and searches in Boulder used programs from the Delila system (10, 11). The search program has been modified to allow searches for relationships between bases, such as complementarity (12). Sequence manipulations and searches in Paris used data bases from the CITI-2 facility.Thermal Denaturation of RNA Hairpins. Experiments were conducted in 0.1 M NaCl/10 mM Na2HPO4/0.1 mM EDTA, pH 7.0. The absorbance was measured at 240 nm. Absorbance-temperature profiles were independent of RNA concentration between 1 and 20 ,uM, indicating that hairpin loops rather than dimers had formed. This was confirmed by showing that the RNA oligomers migrated as monomers on an Altex (Berkeley, CA) Spherogel-TSK exclusion column run at 25°C in the same buffer. The thermal denaturation experiments were run in 0.1 M NaCl buffer because the transition hyperchromic shift occurred at an indeterminably high temperature for the CUUCGG hairpin when the experiments were conducted in 1 M NaCl. The absorbance values were used to extrapolate upper and lower baselines from which were calculated the fraction of helix versus temperature as described by Uhlenbeck et al. (13). RESULTS The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. CTTCGG Predicts

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“…AUU gives poor toeprints with initiator tRNA fragments, and IF3 destabilizes them. Furthermore, in vivo translation from an AUA codon is very poor (Shinedling et al 1987). The guanosine in the third position of the initiation codon is probably essential for IF3 selection of initiator tRNA.…”

Section: Role Of the Initiation Codonmentioning

confidence: 99%

Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3.

Hartz¹,

Binkley²,

et al. 1990

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Initiation factors are used by Escherichia cob to select the initiator tRNA over elongator tRNAs during translation initiation. IF3 appears to "inspect" the anticodon end of the tRNA, probably along with the initiation codon. The anticodon stem and loop of the initiator tRNA, together with part of the initiation codon of the mRNA, can be thought of as a unit. Changes made in the anticodon stem, the anticodon loop, or the anticodon of an initiator tRNA fragment result in a loss of selection by IF3 in an in vitro assay for translation initiation. IF3 allows the selection of an initiator tRNA anticodon stem and loop fragment on GUG and UUG codons but does not select that tRNA fragment in response to AUU. Escherichia coli initiation factor IF3 is involved in several aspects of translation initiation; IF3 (together with IF1) helps in the dissociation of 70S ribosomes into 30S and 50S subunits (Kaempfer 1972;Godefroy-Colburn et al. 1975), catalyzes the formation of 30S initiation complexes (Wintermeyer and Gualerzi 1983), and selects the initiator tRNA against elongator tRNAs in those complexes . The selection of the initiator tRNA by IF3 is based on the unique RNA part of the initiator tRNA (Risuleo et al. 1976), and, most likely, sequences located in the anticodon stem and loop . The three GC base pairs of the anticodon stem adjacent to the loop of the initiator tRNA are important for initiation complex formation and in vitro translation (Seong and RajBhandary 1987), suggesting that this part of the anticodon stem might be important for recognition by IF3. Berkhout et al. (1986) showed that IF3 excludes initiator tRNA on a UUU codon, as though the initiation codon, itself, might be part of the recognition motif explored by IF3 during selection of the initiator tRNA.We have examined those features of the initiator tRNA that are required for selection by IF3. Using toeprinting (Winter et al. 1987;Hartz et al. 1988Hartz et al. , 1989McPheeters et al. 1988;Blasi et al. 1989;Schaefer et al. 1989) as the assay for tRNA binding in 30S initiation complexes, we tested initiator tRNA fragments and several mutant fragments for their ability to be selected by IF3 on various initiation codons. ResultsTo analyze 30S complex formation on various translation initiation sites, we used the extension inhibition technique, also called toeprinting, cDNA synthesis on a template mRNA is terminated when the reverse transcriptase encounters a 30S ribosomal subunit plus tRNA bound on the mRNA. The short cDNA is visualized as a toeprint band on a sequencing gel. The toeprint usually appears 15 bases downstream of the A of the initiation codon if initiator tRNA is bound in the complex or at + 15 from the first base of the cognate codon if elongator tRNA is bound. When compared to the band of the fulllength cDNA, the toeprint band is a quantitative measure of ternary complex formation . Variations of A22~ etUsing the toeprinting assay, we have shown previously that the synthetic initiator tRNA fragment A22~ et (Fig. 1) behaves very similarly ...

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Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Cited by 49 publications

References 40 publications

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene

CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.

Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3.

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