The 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A.... Show moreThe 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5' UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops, Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants, Different symptoms were observed for the various mutants, pointing to a functional role of the C-C and C-A mismatches in the hairpins of the 5' UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants, Remarkably, in all cases C-C or C-A mismatches were introduced. The internal loop of the 5'-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin, A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed. Show less
Rossum, B.J. van; Wachtveitl, J.; Raap, J.; Hoef, K. van der; Gast, P.; Lugtenburg, J.; ... ; Groot, H.J.M. de 1997
CP/MAS NMR data collected from L162YL mutant [4'-C-13]Tyr-enriched Rhodobacter sphaeroides RCs reveal that Tyr L162 is in a slightly heterogeneous and probably rigid section of the protein complex.... Show moreCP/MAS NMR data collected from L162YL mutant [4'-C-13]Tyr-enriched Rhodobacter sphaeroides RCs reveal that Tyr L162 is in a slightly heterogeneous and probably rigid section of the protein complex. The differences in chemical shifts of the individual components relative to those of the [4'-C-13]Tyr Rhodobacter sphaeroides R26 response are 0.2 ppm or less. This is small compared to the total dispersion of [4'-C-13] isotropic shifts, similar to 5 ppm, which measures the shift range due to variations in the microscopic environment between the various tyrosines in the protein complex. The structural changes in the mutant with respect to Rhodobacter sphaeroides R26, as probed by the labels, are thus minimal on the scale of the NMR. This suggests that the dramatic decrease of re-reduction rate of the oxidized primary donor P upon mutation (Farchaus et al., Biochemistry 32 (1993) 10885-10893) cannot be attributed to significant structural changes in the protein. Hence the NMR is in line with the current view that the decrease of the re-reduction rate in the mutant originates from slow reorientation of the docked cytochrome. (C) 1997 Elsevier Science B.V. Show less
By selective isotope enrichment of astaxanthin, MAS NMR and semi-empirical modelling, ligand-protein interactions associated with the red shift in alpha-crustacyanin, the major blue astaxanthin... Show moreBy selective isotope enrichment of astaxanthin, MAS NMR and semi-empirical modelling, ligand-protein interactions associated with the red shift in alpha-crustacyanin, the major blue astaxanthin binding carotenoprotein complex from the carapace of the lobster Homarus gammarus, have been analysed. C-13 Magic Angle Spinning (MAS) NMR spectra were obtained after reconstitution with astaxanthins labelled in the centre of the molecule or at the two keto groups. The MAS data reveal electrostatic polarizations of the conjugated chain. In addition, solid state NMR results for pure unlabelled astaxanthin can be compared with natural abundance C-13 MAS data for canthaxanthin and beta-carotene, to address the effect of the ring functionalities on the electronic properties of the polyene chain. Quantum chemical calculations were performed to reconcile the MAS data with one of several simple and straightforward mechanisms for the colour shift. The results point towards a colour shift mechanism in which the astaxanthin may be doubly charged, possibly by a double protonation of the two ring keto groups. Show less
The tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro.... Show moreThe tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro. Deletions in the 5'-to 3' direction up to the pseudoknot structure did not result in a decrease of transcription efficiency. However, transcription efficiency was reduced twofold when a fragment of 21 nucleotides, comprising the 3'-terminal hairpin, was used as a template, tRNA(Phe) from yeast, Escherichia coli 5S rRNA, and the 3'-terminal 208 nucleotides of alfalfa mosaic virus RNA 3 could not be transcribed by the RNA-dependent RNA polymerase (RdRp) of TYMV. Various mutations in the sequences of loop regions L1 and L2 or of stem region S1 of the pseudoknot were tested to further investigate the importance of the pseudoknot structure. The results were compared with those obtained in an earlier study on aminoacylation with the same mutants tR, M. W. Mans, M. H. van Steeg, P. W. G. Verlaan, C. W. A. Pleij, and L. Bosch. J. Mol. Biol, 223:221-232: 1992). Mutants which still harbor a stable pseudoknot, as proven by probing its structure, have a transcription efficiency very close to that of the wild-type virus. Disruption of the pseudoknot structure, however, gives rise to a drop in transcription efficiency to about 50%, No indications of base-specific interactions between L1, L2, or S1 of the pseudoknot and the RdRp were found. Show less
We report that tyro subtypes of alpha(2)-adrenergic receptors (alpha(2A/D)- and alpha(2C)-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a... Show moreWe report that tyro subtypes of alpha(2)-adrenergic receptors (alpha(2A/D)- and alpha(2C)-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha(2C)-AR mRNA that impairs translational processing, NIH-3T3 fibroblasts mere transfected with receptor constructs (coding region plus 552 nt, alpha(2C)-AR; coding region plus 1140 nt, alpha(2A/D)-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein, 90% of the NIH-3T3 alpha(2C)-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell Lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha(2A/D)-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha(2C)-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR. was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha(2C)-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a hey mechanism for regulating alpha(2C)-AR expression and associated signaling events. Show less
Egorova-Zachernyuk, A.; Rossum, B. van; Boender, G.J.; Franken, E.; Ashurst, J.; Raap, J.; ... ; Groot, H.J.M. de 1997
The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have... Show moreThe electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from C-13 chemical shift data, A new experimental approach involving multispin C-13 labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly C-13 biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state C-13 magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state C-13-C-13 correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many C-13 resonances of the [U-C-13]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of C-13 shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer. Show less
The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the... Show moreThe RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3' terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion With ribonuclease T1. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66. (C) 1997 Elsevier Science B.V. Show less
Poot, R.A.; Jeeninga, R.E.; Pleij, C.W.A.; Duin, J. van 1997
We have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit... Show moreWe have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit translational activity in vivo and to affect ribosome stability in vitro, The majority of the mutant 30S particles were present as free subunits in which a reproducible decrease in amount of proteins S1, S2, S18 and S21 was observed, The protein gels also showed the appearance of a satellite band nest to S5, This band reacted with anti-S5 antibodies and had a slightly increased positive charge, The simplest interpretation of these findings, also considering published data, is that the satellite band is S5 with a non-acetylated N-terminal alanine, Underacetylation of S5 due to mutations in the 16S rRNA implies that the modification is performed on the ribosome. Show less
Verdegem, P.J.E.; Helmle, M.; Lugtenburg, J.; Groot, H.J.M. de 1997
The results presented in this paper show that accurate through-space internuclear distance measurements can be performed on doubly labeled retinals using the one-dimensional approach to the solid... Show moreThe results presented in this paper show that accurate through-space internuclear distance measurements can be performed on doubly labeled retinals using the one-dimensional approach to the solid state magic angle spinning (MAS) rotational resonance NMR technique. The apparent splitting Delta omega(1) of the resonances at n = 1 rotational resonance for the labeled vinylic positions of (all-E)-[10,20-C-13(2)]retinal, (all-E)-[11,20-C-13(2)]retinal, and (all-E)-[12,20-C-13(2)]retinal can be simulated with a coherent set of parameters. From a series of simulations with different dipolar coupling constant brs, it appears that b(IS)/2 pi root 8 = 1.15(Delta omega(1)/2 pi) + 7 (Hz) to a good approximation. Using this relationship as a calibration, it is demonstrated with a set of model compounds that straightforward Lorentzian fitting to measure Delta omega(1) can be used to determine internuclear distances up to 0.44 nm in doubly labeled retinals in the solid state. Show less
The pseudoknot or "base-paired loop region" is a widespread structural motif in all kinds of viral RNAs. Detailed structures of hairpin-type pseudoknots, obtained by NMR, are now emerging, but it... Show moreThe pseudoknot or "base-paired loop region" is a widespread structural motif in all kinds of viral RNAs. Detailed structures of hairpin-type pseudoknots, obtained by NMR, are now emerging, but it is still not clear which structural features are responsible for the different functions in processes like translation and replication. Especially noncoding regions are rich sources of pseudoknot structures, where they occur in domains like IRES elements and tRNA-like structures. But also its role in coding regions like in ribosomal -1 frameshifting and read-through is well established, although the precise mechanism of interference with the translational mechanism remains unknown. (C) 1997 Academic Press. Show less
Kolbert, A.C.; Verel, R.; Groot, H.J.M. de; Almeida, M. 1997
