The structure of clathrate hydrates with tetraisoamylammonium polyacrylate salt incorporated as guest has been studied in this work. Also, quantitative studies on the stability changes of the ...clathrate hydrates with different degrees of cross-linking of the guest polymer (varied from 0 to 3%) have been conducted. A single crystal X-ray diffraction study of a crystal of the hydrate with linear (uncross-linked) tetraisoamylammonium polyacrylate as guest reveals a hexagonal structure (space group P6̅m2, a = 12.15 Å, c =12.58 Å at 100 K) with 39 host framework water molecules per one guest monomeric unit. Powder X-ray diffraction analyses confirm the identity of the above crystal structure of the hydrate with linear guest polymer and the crystal structure of the hydrates with cross-linked guest (hexagonal, a = 12.25 Å, c =12.72 Å at 276 K). In order to quantitatively determine the stability differences of the hydrates with the included guests having various degrees of cross-linking of the anionic chain, a series of differential scanning calorimetry measurements of the fusion enthalpy of the hydrate samples has been carried out. On the basis of the results obtained, a structural model describing the decrease in the stability of the clathrate hydrates with tetraisoamylammonium polyacrylate guest as a function of the degree of cross-linking of the guest polymer has been suggested.
The 18S rRNA environment of the mRNA at the decoding
site of human 80S ribosomes has been studied by crosslinking
with derivatives of hexaribonucleotide UUUGUU (comprising
Phe and Val codons) that ...carried a perfluorophenylazide
group either at the N7 atom of the guanine or at the C5
atom of the 5′-terminal uracil residue. The location
of the codons on the ribosome at A, P, or E sites has been
adjusted by the cognate tRNAs. Three types of complexes
have been obtained for each type derivative, namely, (1)
codon UUU and Phe-tRNAPhe at the P site (codon
GUU at the A site), (2) codon UUU and tRNAPhe
at the P site and PheVal-tRNAVal at the A site,
and (3) codon GUU and Val-tRNAVal at the P site
(codon UUU at the E site). This allowed the placement of
modified nucleotides of the mRNA analog at positions −3,
+1, or +4 on the ribosome. Mild UV irradiation resulted
in tRNA-dependent crosslinking of the mRNA analogs to the
18S rRNA. Nucleotide G961 crosslinked to mRNA position
−3, nucleotide G1207 to position +1, and A1823 together
with A1824 to position +4. All of these nucleotides are
located in the most strongly conserved regions of the small
subunit RNA structure, and correspond to nucleotides G693,
G926, G1491, and A1492 of bacterial 16S rRNA. Three of
them (with the exception of G1491) had been found earlier
at the 70S ribosomal decoding site. The similarities and
differences between the 16S and 18S rRNA decoding sites
are discussed.
The cDNA of human ribosomal protein S13 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in
Escherichia coli cells. Protein accumulated ...in the form of inclusion bodies was isolated, purified, and refolded by dialysis. The recombinant protein was immunologically reactive, interacting with antiserum against native rpS13. The secondary structure content of the refolded protein was analyzed by means of CD spectroscopy. It was found that 43±5% of amino acids sequence of the protein form α-helices and 11±3% are placed in β-strands that coincides with theoretical predictions. The β-strands seem to be located in the extension regions of the rpS13 and do not have homologuous regions in the structure of rpS15 from
Thermus thermophilus, which is a prokaryotic homolog of rpS13. The protein structure is stable at a pH range from 4.0 to 8.0 and at low concentrations of urea (up to 3 M).
A sequence-specific modification of the human 5.8 S rRNA in isolated 60 S subunits, non-programmed 80 S ribosomes and ribosomes complexed with mRNA and tRNAs was studied with the use of a derivative ...of the nonaribonucleotide UCUGUGUUU bearing a perfluorophenylazide group on the C-5 atom of the 5'-terminal uridine. Part of the oligonucleotide moiety of the derivative was complementary to the 5.8 S rRNA sequence ACACA in positions 82-86 flanked by two guanines at the 5'-terminus. The target for the cross-linking was identified as nucleotide G89 on the 5.8 S RNA. In addition, several ribosomal proteins were modified by the oligonucleotide derivative bound to the 5.8 S rRNA and proteins L6 and L8 were among them. Application of these results to known cryo-electron microscopy images of eukaryotic 60 S subunits made it possible to suggest that the central part of the 5.8 S rRNA containing the sequence 82-86 and proteins L6 and L8 are located at the base of the L1 stalk of the 60 S subunit. The efficacy of cross-linking in non-programmed 80 S ribosomes was much lower than in isolated 60 S subunits and in programmed 80 S ribosomes. We suggest that the difference in the accessibilities of the central part of the 5.8 S rRNA in the programmed and non-programmed 80 S ribosomes is caused by a conformational switch that seems to be required to dissociate the 80 S ribosomes into the subunits after termination of translation to allow initiation of translation of a new template.
Positioning of each nucleotide of the E site and the P site bound codons with respect to the 18S rRNA on the human ribosome was studied by cross-linking with mRNA analogs, derivatives of the ...hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group on the second or the third uracil, and a derivative of the dodecaribonucleotide UUAGUAUUUAUU with a similar group on the guanine residue. The location of the modified nucleotides at any mRNA position from −3 to +3 (position +1 corresponds to the 5′ nucleotide of the P site bound codon) was adjusted by the cognate tRNAs. A modified uridine at positions from −1 to +3 cross-linked to nucleotide G1207 of the 18S rRNA, and to nucleotide G961 when it was in position −2. A modified guanosine cross-linked to nucleotide G1207 if it was in position −3 of the mRNA. These data indicate that nucleotide G961 of the 18S rRNA is close only to mRNA positions −3 and −2, while G1207 is in the vicinity of positions from −3 to +3. The latter suggests that there is a sharp turn between the P and E site bound codons that brings nucleotide G1207 of the 18S rRNA close to each nucleotide of these codons. This correlates well with X-ray crystallographic data on bacterial ribosomes, indicating existence of a sharp turn between the P site and E site bound codons near a conserved nucleotide G926 of the 16S rRNA (corresponding to G1207 in 18S rRNA) close to helix 23b containing the conserved nucleotide 693 of the 16S rRNA (corresponding exactly to G961 of the 18S rRNA).
Hybridization of two oligodeoxyribonucleotides (ON1 and ON2), complementary to opposite strands of the apical domain of
Escherichia coli 4.5S RNA, was studied. ON1, complementary to bases 58–71, was ...not able to form a stable RNA-DNA hybrid whereas ON2, complementary to bases 38–53, was. Addition of both oligonucleotides at the same time resulted in the formation of a ternary complex permitting hybridization of ON1 and increasing hybridization of ON2. Under this condition, binary complexes of ON1 or ON2 with 4.5S RNA were not observed. The data demonstrate that hybridization of oligonucleotides to both strands of an RNA hairpin structure increases the efficiency of hybridization of either oligonucleotide.
Site‐specific alkylation of RNA by reactive oligodeoxynucleotides provides structural information and represents the first step towards the design of RNA derivatives to be used for functional ...studies. Specific alkylation of 4.5S RNA at G53, the first base of the apical tetraloop, was achieved by incubation with oligodeoxynucleotide ON2, complementary to nucleotides 38−53, which carries a p‐(N‐2‐chloroethyl‐N‐methylamino)benzylamidophosphate group at the 5′ end. Alkylation efficiency was increased by a factor of 6, without alteration of specificity, in the presence of a helper oligodeoxynucleotide, ON1, complementary to nucleotides 58−71 of the opposite strand of the RNA helix. A second reactive oligodeoxynucleotide, ON1‐3′‐R, was obtained by attaching the alkylating group to the 3′ end of ON1. ON1‐3′‐R was able to modify G58. In the presence of ON2 as a helper oligodeoxynucleotide, the specificity of ON1‐3′‐R changes and efficient alkylation of nucleotides G54, A56 and G57 of the apical region of 4.5S RNA was observed.
We describe a methodology which allows the introduction of a photoactivatable azido group at specific internal positions of any RNA in order to identify the neighboring elements of an interacting ...protein. The first step involves site-directed modification of the target RNA with an antisense oligodeoxyribonucleotide bearing, at its 3' or 5' phosphate, a 4--N-(2-chloroethyl)-N-methylaminobenzylmethylamino group. Position N7 of a guanine residue located in the close vicinity of the hybrid is the main target for alkylation. The antisense oligodeoxyribonucleotide is then removed by acidic pH treatment and a photoreactive reagent (2,4-dinitro-5-fluorophenylazide) is condensed to the modified nucleotide. This method was used to induce specific cross-links between Escherichia coli threonyl-tRNA synthetase and the leader region of threonyl-tRNA synthetase mRNA, which is involved in translational feedback regulation. Control experiments revealed that the modification affects neither the structure of the mRNA nor the interaction with the enzyme. More than 50% of the modified mRNA complexed with threonyl-tRNA synthetase can be cross-linked to the enzyme, depending on the nucleotide modified.
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