Biomedical subjects

D Konings

Publications and source records attributed to D Konings.

8 recordsLinked to original sources

PubMed2001

Recognition of peroxisomal targeting signal type 1 by the import receptor Pex5p.

We have studied how Pex5p recognizes peroxisomal targeting signal type 1 (PTS1)-containing proteins. A randomly mutagenized pex5 library was screened in a two-hybrid setup for mutations that disrupted the interaction with the PTS1 protein Mdh3p or for suppressor mutations that could restore the interaction with Mdh3p containing a mutation in its PTS1. All mutations localized in the tetratricopeptide repeat (TPR) domain of Pex5p. The Pex5p TPR domain was modeled based on the crystal structure of a related TPR protein. Mapping of the mutations on this structural model revealed that some of the loss-of-interaction mutations consisted of substitutions in alpha-helices of TPRs with bulky amino acids, probably resulting in local misfolding and thereby indirectly preventing binding of PTS1 proteins. The other loss-of-interaction mutations and most suppressor mutations localized in short, exposed, intra-repeat loops of TPR2, TPR3, and TPR6, which are predicted to mediate direct interaction with PTS1 amino acids. Additional site-directed mutants at conserved positions in intra-repeat loops underscored the importance of the loops of TPR2 and TPR3 for PTS1 interaction. Based on the mutational analysis and the structural model, we put forward a model as to how PTS1 proteins are selected by Pex5p.

Amino Acid Sequence↗

PubMed2000

Predicting U-turns in ribosomal RNA with comparative sequence analysis.

The U-turn is a well-known RNA motif characterized by a sharp reversal of the RNA backbone following a single-stranded uridine base. In experimentally determined U-turn motifs, the nucleotides 3' to the turn are frequently involved in tertiary interactions, rendering this motif particularly attractive in RNA modeling and functional studies. The U-turn signature is composed of an UNR sequence pattern flanked by a Y:Y, Y:A (Y=pyrimidine) or G:A base juxtaposition. We have identified 33 potential UNR-type U-turns and 25 related GNRA-type U-turns in a large set of aligned 16 S and 23 S rRNA sequences. U-turn candidates occur in hairpin loops (34 times) as well as in internal and multi-stem loops (24 times). These are classified into ten families based on loop type, sequence pattern (UNR or GNRA) and the nature of the closing base juxtaposition. In 13 cases, the bases on the 3' side of the turn, or on the immediate 5' side, are involved in tertiary covariations, making these sites strong candidates for tertiary interactions.

Animals↗

PubMed1997

Assessing the reliability of RNA folding using statistical mechanics.

We have analyzed the base-pairing probability distributions of 16 S and 16 S-like, and 23 S and 23 S-like ribosomal RNAs of Archaea, Bacteria, chloroplasts, mitochondria and Eukarya, as predicted by the partition function approach for RNA folding introduced by McCaskill. A quantitative analysis of the reliability of RNA folding is done by comparing the base-pairing probability distributions with the structures predicted by comparative sequence analysis (comparative structures). We distinguish two factors that show a relationship to the reliability of RNA minimum free energy structure. The first factor is the dominance of one particular base-pair or the absence of base-pairing for a given base within the base-pairing probability distribution (BPPD). We characterize the BPPD per base, including the probability of not base-pairing, by its Shannon entropy (S). The S value indicates the uncertainty about the base-pairing of a base: low S values result from BPPDs that are strongly dominated by a single base-pair or by the absence of base-pairing. We show that bases with low S values have a relatively high probability that their minimum free energy (MFE) structure corresponds to the comparative structure. The BPPDs of prokaryotes that live at high temperatures (thermophilic Archaea and Bacteria) have, calculated at 37 degrees C, lower S values than the BPPDs of prokaryotes that live at lower temperatures (mesophilic and psychrophilic Archaea and Bacteria). This reflects an adaptation of the ribosomal RNAs to the environmental temperature. A second factor that is important to consider with regard to the reliability of MFE structure folding is a variable degree of applicability of the thermodynamic model of RNA folding for different groups of RNAs. Here we show that among the bases that show low S values, the Archaea and Bacteria have similar, high probabilities (0.96 and 0.94 in 16 S and 0.93 and 0.91 in 23 S, respectively) that the MFE structure corresponds to the comparative structure. These probabilities are lower in the chloroplasts (16 S 0.91, 23 S 0.79), mitochondria (16 S-like 0.89, 23 S-like 0.69) and Eukarya (18 S 0.81, 28 S 0.86).

Computer Simulation↗

PubMed1995

G.U base pairing motifs in ribosomal RNA.

An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs. Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems. In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5'UG3' 3'GA5' and 5'UG3' 3'GU5', as well as G.U pairs flanked by a bulge loop 3' of U. The possible structures and functions of these recurrent motifs are discussed.

Base Composition↗

PubMed1994

A major family of motifs involving G.A mismatches in ribosomal RNA.

G.A oppositions and their flanking nucleotides in the internal loops of 16 S and 23 S rRNA were analyzed from a comparative structure perspective, resulting in an unexpectedly high incidence of the sequence motifs [formula: see text], [formula: see text] and [formula: see text]. The first two motifs can form similar three-dimensional structures containing sheared G.A or A.A pair conformations. Comparative sequence analysis revealed numerous sites in ribosomal RNAs with distinct combinations of nucleotides capable of forming this specific structure. In some cases, the sequence variations provide strong evidence for the sheared tandem structure occurring. Interestingly, the sequence changes would maintain a similar exposure of two adenines in the minor groove, suggesting the possibility that they are serving as a recognition or anchoring unit. These tandem conformations are related to that of the [formula: see text] tandem observed in 5 S and 28 S rRNA.

Hydrogen Bonding↗

PubMed1989

Structure-function relationships in a self-splicing group II intron: a large part of domain II of the mitochondrial intron aI5 is not essential for self-splicing.

An oligonucleotide-directed deletion of 156 nucleotides has been introduced into the yeast mitochondrial group II intron al5 (887 nt). The deletion comprises almost all of domain II, which is one of the six phylogenetically conserved structural elements of group II introns. This mutant displays reduced self-splicing activity, but results of chemical probing with dimethylsulphate suggest that sequences at the site of the deletion interfere with the normal folding of the intron. This is supported by computer analyses, which predict a number of alternative structures involving conserved intron sequences. Splicing activity could be restored by insertion of a 10-nucleotide palindromic sequence into the unique Smal site of the deletion mutant, resulting in the formation of a small stable stem-loop element at the position of domain II. These results provide a direct correlation between folding of the RNA and its activity. We conclude that at least a large part of domain II of the group II intron al5 is not required for self-splicing activity. This deletion mutant with a length of 731 nucleotides represents the smallest self-splicing group II intron so far known.

Base Sequence↗

PubMed1988

Secondary structure and expression in vivo and in vitro of messenger RNAs into which upstream AUG codons have been inserted.

We wanted to discover whether the conformation of the mRNA leader sequence is involved in translational fidelity. For this purpose we constructed several mutants of Semliki Forest virus 26S mRNA and inserted AUG codons into the leader sequence. We then analyzed the results of in vitro and in vivo translation of these mRNAs, probed enzymatically the secondary structure and performed minimal energy folding of the transcripts. Our results indicate that the position of a hairpin in the leader sequence determines at which AUG codon downstream from that hairpin translation is initiated.

Base Sequence↗

PubMedDate not supplied

Familial Alzheimer's mutation: mRNA secondary structure revisited.

It has been suggested that the mutation at position 717 of the amyloid precursor protein (APP), found in several cases of familial Alzheimer's disease, affects the secondary structure of the corresponding messenger RNA and the rate of its translation (10). Phylogenetic analysis based on comparison with other mammalian APP sequences does not support this possibility.

Alzheimer Disease↗