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Title: Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.

Abstract

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of themore » CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.« less

Authors:
 [1];  [2];  [1];  [3];  [4]
  1. (Washington State University)
  2. (ASSOC WESTERN UNIVERSITY)
  3. (BATTELLE (PACIFIC NW LAB))
  4. (VISITORS)
Publication Date:
Research Org.:
Pacific Northwest National Lab., Richland, WA (US)
Sponsoring Org.:
US Department of Energy (US)
OSTI Identifier:
15003117
Report Number(s):
PNWD-SA-5349
TRN: US200420%%356
DOE Contract Number:
AC06-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 39; Journal Issue: 3; Other Information: PBD: 25 Jan 2000
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CHROMATIN; DIGESTION; DISTRIBUTION; DNA; ENZYMES; GENES; HISTONES; HYDROXYL RADICALS; INDUCTION; MODULATION; NUCLEASES; NUCLEOSOMES; PYRIMIDINE DIMERS; PYRIMIDINES; RIBOSOMAL RNA

Citation Formats

Liu, X, Mann, David B., Suquet, C, Springer, David L., and Smerdon, Michael J. Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.. United States: N. p., 2000. Web. doi:10.1021/bi991771m.
Liu, X, Mann, David B., Suquet, C, Springer, David L., & Smerdon, Michael J. Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.. United States. doi:10.1021/bi991771m.
Liu, X, Mann, David B., Suquet, C, Springer, David L., and Smerdon, Michael J. Tue . "Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.". United States. doi:10.1021/bi991771m.
@article{osti_15003117,
title = {Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.},
author = {Liu, X and Mann, David B. and Suquet, C and Springer, David L. and Smerdon, Michael J.},
abstractNote = {The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.},
doi = {10.1021/bi991771m},
journal = {Biochemistry},
number = 3,
volume = 39,
place = {United States},
year = {Tue Jan 25 00:00:00 EST 2000},
month = {Tue Jan 25 00:00:00 EST 2000}
}
  • Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1. Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure. All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G{sub 80}{center dot}C{sub 92}{center dot}G{sub 81}{center dot}C{sub 91}-G{sub 82}{center dot}C{sub 90}-A{sub 83}{center dot}U{sub 89}-C{sub 84}{center dot}G{sub 88} and three unpaired U's in helix IV by proton homonuclear Overhauser enhancementmore » connectivities. The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA. In addition, G{sub 21}{center dot}C{sub 58}-A{sub 20}{center dot}U{sub 59}-G{sub 19}{center dot}C{sub 60}-A{sub 18}{center dot}U{sub 61} in helix II, U{sub 32}{center dot}A{sub 46}-G{sub 31}{center dot}C{sub 47}-C{sub 30}{center dot}G{sub 48}-C{sub 29}{center dot}G{sub 49} in helix III, and G{sub 4}{center dot}C{sub 112}-G{sub 5}{center dot}C{sub 111}-U{sub 6}{center dot}G{sub 110} in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C. Base pairs in helices I-IV of the universal secondary structure of B. megaterium 5S RNA are described.« less
  • The precise molecular composition of the Xenopus laevis TFIIIA-5S ribosomal RNA complex (7S particle) has been established from small angle neutron and dynamic light scattering. The molecular weight of the particle was found to be 95,700{plus minus}10,000 and 86,700{plus minus}9,000 daltons from these two methods respectively. The observed match point of 54.4% D{sub 2}O obtained from contrast variation experiments indicates a 1:1 molar ratio. It is concluded that only a single molecule of TFIIIA, a zinc-finger protein, and of 5S RNA are present in this complex. A simple elongated cylindrical model with dimensions of 140 {angstrom} length and 59 {angstrom}more » diameter is compatible with the neutron results. A globular model can be excluded by the shallow nature of the neutron scattering curves. It is proposed that the observed difference of 15 {angstrom} in length between the 7S particle and isolated 5S RNA most likely indicates that part(s) of the protein protrudes from the end(s) of the RNA molecule. There is no biochemical evidence for any gross alteration in 5S RNA conformation upon binding to TFIIIA.« less
  • The base-pair protons of the common arm duplex fragment of wheat germ (Triticum aestivum) ribosomal 5S RNA have been identified and assigned by means of 500-MHz proton NMR spectroscopy. The two previously reported extra base pairs within the fragment are now explained by the presence of two distinct solution structures of the common arm fragment (and its corresponding base-paired segment in intact 5S rRNA). The present conclusions are supported by one- and two-dimensional proton homonuclear Overhauser enhancements in H{sub 2}O and by temperature variation and Mg{sup 2+} titration of the downfield {sup 1}H NMR spectrum. The difference between the twomore » conformers is most likely due to difference in helical tightness. Some additional amino proton resonances have also been assigned.« less
  • The nonexchangeable protons of the common arm fragment of wheat germ (Triticum aestivum) ribosomal 5S RNA have been observed by means of high-resolution 500-MHz {sup 1}H NMR spectroscopy in D{sub 2}O solution. Although NMR studies on the exchangeable protons support the presence of two distinct solution structures of the common arm fragment (and of the same base-paired segment in intact 5S rRNA), only a single conformation is manifested in the {sup 1}H NMR behavior of all of the H6 and H5 pyrimidine and most of the H8/H2 purine protons under the same salt conditions. The nonexchangeable protons near the base-pairedmore » helix have been assigned by a sequential strategy. Conformational features such as the presence of a cytidine-uridine (C{center dot}U) pair at the loop-helix junction and base stacking into the hairpin loop are evaluated from nuclear Overhauser enhancement spectroscopy (NOESY) data. Double-quantum filtered correlation spectroscopy (DQF-COSY) experiments show that most of the 26 riboses are in the C3{prime}-endo conformation. Finally, backbone conformational changes induced by Mg{sup 2+} and heating have been monitored by {sup 31}P NMR spectroscopy. The results show that the common arm RNA segment can assume two conformations which produce distinguishably different NMR environments at the base-pair hydrogen-bond imino protons but not at nonexchangeable base or ribose proton or backbone phosphate sites.« less