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J F Atkins

Publications and source records attributed to J F Atkins.

At least 37 records · Page 2Linked to original sources
PubMed2000

The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit.

Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.

Binding Sites↗
PubMed2000

Nonlinearity in genetic decoding: homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting.

The tau and gamma subunits of DNA polymerase III are both encoded by a single gene in Escherichia coli and Thermus thermophilus. gamma is two-thirds the size of tau and shares virtually all its amino acid sequence with tau. E. coli and T. thermophilus have evolved very different mechanisms for setting the approximate 1:1 ratio between tau and gamma. Both mechanisms put ribosomes into alternate reading frames so that stop codons in the new frame serve to make the smaller gamma protein. In E. coli, approximately 50% of initiating ribosomes translate the dnaX mRNA conventionally to give tau, but the other 50% shift into the -1 reading frame at a specific site (A AAA AAG) in the mRNA to produce gamma. In T. thermophilus ribosomal frameshifting is not required: the dnaX mRNA is a heterogeneous population of molecules with different numbers of A residues arising from transcriptional slippage on a run of nine T residues in the DNA template. Translation of the subpopulation containing nine As (or +/- multiples of three As) yields tau. The rest of the population of mRNAs (containing nine +/- nonmultiples of three As) puts ribosomes into the alternate reading frames to produce the gamma protein(s). It is surprising that two rather similar dnaX sequences in E. coli and T. thermophilus lead to very different mechanisms of expression.

Bacterial Proteins↗
PubMed2000

ODNBase--a web database for antisense oligonucleotide effectiveness studies. Oligodeoxynucleotides.

SUMMARY: ODNBase is a database of antisense oligodeoxynucleotides targeted to mammalian mRNAs that were reported in the literature. It includes the oligo sequences tested, the measured effectiveness, the RNA that was targeted, the type of measurement assay used, the oligo concentration applied, and the reference for each oligo. It provides a searchable interface by motif content, activity level, applied concentration and RNA name. Oligo lists matching search criteria can be downloaded in a spreadsheet compatible format.

Animals↗
PubMed2000

Coupling of open reading frames by translational bypassing.

Translational bypassing joins the information found within two disparate open reading frames into a single polypeptide chain. The underlying mechanism centers on the decoding properties of peptidyl-transfer RNA (tRNA) and involves three stages: take-off, scanning, and landing. In take-off, the peptidyl-tRNA/messenger RNA (mRNA) complex in the P site of the ribosome dissociates, and the mRNA begins to move through the ribosome. In scanning, the peptidyl-tRNA probes the mRNA sliding through the decoding center. In landing, the peptidyl-tRNA re-pairs with a codon with which it can form a stable interaction. Although few examples of genes are known that rely on translational bypassing to couple open reading frames, ribosomes appear to have an innate capacity for bypassing. This suggests that the strategy of translational bypassing may be more common than presently appreciated. The best characterized example of this phenomenon is T4 gene 60, in which a complex set of signals stimulates bypassing of 50 nucleotides between the two open reading frames. In this review, we focus on the bypassing mechanism of gene 60 in terms of take-off, scanning, and landing.

Amino Acid Sequence↗
PubMed2000

Sequence specificity of aminoglycoside-induced stop condon readthrough: potential implications for treatment of Duchenne muscular dystrophy.

As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside-induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside-induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside-induced readthrough in the order C > U > A > or = G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33-base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low-level aminoglycoside-induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed.

Animals↗
PubMed1999

Eubacterial tmRNAs: everywhere except the alpha-proteobacteria?

Eubacterial tmRNAs mediate, at least in Escherichia coli, recycling of ribosomes stalled at the end of terminatorless mRNAs. A tmRNA-encoded peptide tag is added to abnormal protein products of truncated mRNAs. This tag is a specific signal for proteolysis of the aberrant protein. To obtain further sequence information, PCR was used to amplify more Eubacterial genes for tmRNA. Fifty-eight new tmDNA sequences including from members of nine additional phyla were determined. Remarkably, tmDNA sequences could be amplified from all species tested apart from those in the alpha-Proteobacteria, raising evolutionary implications.

Bacteria↗
PubMed1999

Structure of human ornithine decarboxylase antizyme 2 gene.

Ornithine decarboxylase antizyme 1, a negative regulator of polyamine levels, acts by destabilizing the first enzyme in the polyamine biosynthetic pathway (ODC) and by inhibiting uptake of polyamines. Recently, a second mammalian antizyme was discovered. Here, we report the genomic organization and chromosomal position of the new human gene, HuAZ2, and make a comparison with its antizyme 1 homologue. Fluorescence in-situ hybridization (FISH) localized the sequences from a bacterial artificial chromosomal (BAC) clone containing HuAZ2 at band 22 of the long arm of chromosome 15. This gene spans at least 15kb and contains five exons. No CAAT or TATA sequences could be identified within 800bp upstream of the translation initiation codon, but the 5'-flanking region contains several putative binding sites for other transcription factors. Transcription initiation of this gene appears to be heterogeneous.

5' Untranslated Regions↗
PubMed1999

Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus.

Retroviruses, such as murine leukemia virus (MuLV), whose gag and pol genes are in the same reading frame but separated by a UAG stop codon, require that 5-10 % of ribosomes decode the UAG as an amino acid and continue translation to synthesize the Gag-Pol fusion polyprotein. A specific pseudoknot located eight nucleotides 3' of the UAG is required for this redefinition of the UAG stop codon. The structural probing and mutagenic analyses presented here provide evidence that loop I of the pseudoknot is one nucleotide, stem II has seven base-pairs, and the nucleotides 3' of stem II are important for function. Stem II is more resistant to single-strand-specific probes than stem I. Sequences upstream of the UAG codon allow formation of two competing structures, a stem-loop and the pseudoknot.

Aldehydes↗
PubMed1999

Mutations which alter the elbow region of tRNA2Gly reduce T4 gene 60 translational bypassing efficiency.

Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full-length protein. Bypassing of the coding gap requires peptidyl-tRNA2Gly detachment from a GGA codon (codon 46) followed by re-pairing at a matching GGA codon just before codon 47. Using negative selection, based on the sacB gene from Bacillus subtilis, Escherichia coli mutants were isolated which reduce bypassing efficiency. All of the mutations are in the gene for tRNA2Gly. Most of the mutations disrupt the hydrogen bonding interactions between the D- and T-loops (G18*psi55 and G19*C56) which stabilize the elbow region in nearly all tRNAs. The lone mutation not in the elbow region destabilizes the anticodon stem at position 40. Previously described Salmonella typhimurium mutants of tRNA2Gly, which reduce the stability of the T-loop, were also tested and found to decrease bypassing efficiency. Each tRNA2Gly mutant is functional in translation (tRNA2Gly is essential), but has a decoding efficiency 10- to 20-fold lower than wild-type. This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon-anticodon stability and bypassing.

Bacteriophage T4↗
PubMed1999

A three-way junction and constituent stem-loops as the stimulator for programmed -1 frameshifting in bacterial insertion sequence IS911.

Several signals are required for the programmed frameshifting in translation of IS911 mRNA. These include a Shine Dalgarno (SD)-like sequence, a slippery sequence of six adenine residues and a guanine residue (A6G) and a 3' secondary structure. The structure of the mRNA containing these elements was investigated using chemical and enzymatic probing. The probing data show that the 3' structure is a three-way junction of stems. The function of the three-way junction was investigated by mutagenesis. Disrupting the stability of the structure greatly affects frameshifting and transposition levels as tested by separate in vivo assays. Structural probing and thermal melting profiles indicate that the disrupted three-way junctions have altered structures.

Aldehydes↗
PubMed1999

Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E. coli tmRNA.

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome. A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed. This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E. coli tmRNA tag-encoded sequence. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants. To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-translation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the second stem of the pseudoknot also effect both functions. The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues. The probing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function. Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation.

Alanine↗
PubMed1999

Ribosomal -1 frameshifting during decoding of Bacillus subtilis cdd occurs at the sequence CGA AAG.

During translation of the Bacillus subtilis cdd gene, encoding cytidine deaminase (CDA), a ribosomal -1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids. The frequency of the frameshift is approximately 16%, and it occurs both when the cdd gene is expressed from a multicopy plasmid in Escherichia coli and when it is expressed from the chromosomal copy in B. subtilis. As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species. The different forms have approximately the same specific activity. The cdd gene was cloned in pUC19 such that the lacZ' gene of the vector followed the cdd gene in the -1 reading frame immediately after the cdd stop codon. By using site-directed mutagenesis of the cdd-lacZ' fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-frame cdd stop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site. The possible function of this frameshift in gene expression is discussed.

Amino Acid Sequence↗
PubMed1998

A second mammalian antizyme: conservation of programmed ribosomal frameshifting.

A second mammalian ornithine decarboxylase antizyme was discovered. The deduced protein sequence of the human antizyme2 is 54% identical and 67% similar to human antizyme1 but 99.5% identical to mouse antizyme2. Polyamine-regulated programmed ribosomal frameshifting is used in decoding antizyme2 mRNA as it is for antizyme1 mRNA. The mRNA signals for the programmed frameshifting are similar in the mRNAs for the two antizymes. However, in the stimulatory pseudoknot 3' of the shift site, while the sequences of the stems are highly conserved, the sequences of the loops are divergent. Functional distinctions between antizymes seem likely, but no distinction in the tissue distribution of human antizyme1 and 2 mRNAs was distinguished, though antizyme2 mRNA is 16-fold less abundant than its antizyme1 counterpart. In addition to the previously characterized human antizyme1 mRNA, a second antizyme1 mRNA with an additional 160 nucleotides at its 3' end was identified, and it has a tissue distribution different from that of the shorter antizyme1 mRNA.

Amino Acid Sequence↗
PubMed1998

Flexibility of the nascent polypeptide chain within the ribosome--contacts from the peptide N-terminus to a specific region of the 30S subunit.

The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887-895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked. The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit. The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the 'ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA.

Amino Acid Sequence↗
PubMed1998

A nickel complex cleaves uridine in folded RNA structures: application to E. coli tmRNA and related engineered molecules.

To gain more insight about Escherichia coli tmRNA structure, NiCR, a square planar macrocyclic nickel (II) complex, was used to probe guanine N7 exposure. On the basis of this additional structural information, a refined secondary structure of the molecule is proposed. In addition to its known specificity for guanine N7, we show here that the chemical probe can also cleave at specific uridine residues. In contrast to the alkaline-labile modification of guanine, the reactivity of NiCR at these uridine residues results in direct strand scission. To better characterize the uridine cleavage sites and assess the importance of the RNA structure for the reaction to occur, smaller RNA molecules derived from one pseudoknot (PK4) of E. coli tmRNA containing two uridine cleavage sites were engineered and probed. It is shown that this pseudoknot can fold by itself in solution and that the expected uridine residues are also cleaved by the nickel complex, suggesting that only a local sequence and/or structural context is required for cleavage. In E. coli tmRNA, the five uridine cleavage sites are located in double-stranded regions. These sites contain a G-U wobble base-pair and a downstream uridine which is cleaved. Using smaller RNAs derived from one stem of PK4, systematic changes in the proposed recognition motif indicate that the G-U pair is required for cleavage. Furthermore, there is no cleavage if the G-U pair is reversed. If the recognition motif is moved within the stem, the cleavage site moves accordingly. Additionally, if the recognition motif is changed such that the G-U pair is flanked by two uridine residues, the reactivity occurs only at the 3' uridine. Radical quenching studies have indicated that sulfate radical, as in the case of guanine oxidation, is involved in uridine oxidation. Although additional studies are required to better characterize the reaction, this paper reports a novel specificity for a chemical probe which may be useful for investigating structural motifs involving G-U pairs in folded RNAs.

Base Composition↗
PubMed1998

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches.

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

Base Composition↗