Monomer−Dimer Equilibrium Constants of RNA in the Dimer Initiation Site of Human Immunodeficiency Virus Type 1†
Identifieur interne : 003839 ( Main/Exploration ); précédent : 003838; suivant : 003840Monomer−Dimer Equilibrium Constants of RNA in the Dimer Initiation Site of Human Immunodeficiency Virus Type 1†
Auteurs : Michael F. Shubsda [États-Unis] ; Mark P. Mcpike [États-Unis] ; Jerry Goodisman [États-Unis] ; James C. Dabrowiak [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 1999.
Descripteurs français
- KwdFr :
- ARN viral (), ARN viral (génétique), ARN viral (métabolisme), Autoradiographie, Concentration osmolaire, Dimérisation, Données de séquences moléculaires, Humains, Initiation de la traduction (génétique), Modèles chimiques, Séquence nucléotidique, Température, VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (), VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (génétique), VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (métabolisme), Électricité statique, Électrophorèse sur gel de polyacrylamide.
- MESH :
- génétique : ARN viral, Initiation de la traduction, VIH-1 (Virus de l'Immunodéficience Humaine de type 1).
- métabolisme : ARN viral, VIH-1 (Virus de l'Immunodéficience Humaine de type 1).
- ARN viral, Autoradiographie, Concentration osmolaire, Dimérisation, Données de séquences moléculaires, Humains, Modèles chimiques, Séquence nucléotidique, Température, VIH-1 (Virus de l'Immunodéficience Humaine de type 1), Électricité statique, Électrophorèse sur gel de polyacrylamide.
English descriptors
- KwdEn :
- Autoradiography, Base Sequence, Dimerization, Electrophoresis, Polyacrylamide Gel, HIV-1 (chemistry), HIV-1 (genetics), HIV-1 (metabolism), Humans, Models, Chemical, Molecular Sequence Data, Osmolar Concentration, Peptide Chain Initiation, Translational (genetics), RNA, Viral (chemistry), RNA, Viral (genetics), RNA, Viral (metabolism), Static Electricity, Temperature.
- MESH :
- chemical , chemistry : RNA, Viral.
- chemistry : HIV-1.
- genetics : HIV-1, Peptide Chain Initiation, Translational, RNA, Viral.
- metabolism : HIV-1, RNA, Viral.
- Autoradiography, Base Sequence, Dimerization, Electrophoresis, Polyacrylamide Gel, Humans, Models, Chemical, Molecular Sequence Data, Osmolar Concentration, Static Electricity, Temperature.
Abstract
The genome of the human immunodeficiency virus (HIV) exists as a dimer of two identical RNA molecules hydrogen bonded to each other near their 5‘ ends. The dimer, known to be important for viral infectivity, is formed by two monomers interacting through a stem−loop structure called the dimer initiation site (DIS). An initially formed intermediate, the “kissing” dimer, is unstable and rearranges to the stable, duplex form. In this report we use nondenaturing polyacrylamide gel electrophoresis to measure the monomer−dimer equilibrium constant of three RNA sequences, 41-, 27-, and 19-mers, located in the DIS of the MAL isolate of HIV-1. Experiments in which the RNA was equilibrated at various temperatures before electrophoresis revealed that interconversion is rapid for all the sequences, so that they reach equilibrium in the loading well of the gel at 5 °C before they enter the gel proper. However, interconversion kinetics in the gel are slow, so autoradiographic spot intensities can be used to measure the amounts of monomer and dimer present when the sample entered the gel. After correction for the amount of RNA added with the radiolabel and dilution of samples in the loading well of the gel, dimerization equilibrium constants were calculated from spot intensities. The calculated values of the dimerization constant K at 5 °C were ∼105, ∼106, and ∼108 M-1 for the 41-, 27-, and 19-mers, respectively, in solutions of ionic strength, I, of about 100 mM. The decrease in K by three orders of magnitude between the 19-mer and 41-mer is due in part to the change in rotational entropy of rodlike molecules on dimerization and in part to the increased conformational entropy of the monomers. As expected, increased ionic strength increases the dimerization constant for all three RNAs. For the 41-mer, however, K has a maximum value at I ∼140 mM. The origin of the decrease in K for higher I is unknown but it may be due to formation of species (perhaps higher order oligomers) that do not enter the gel. The 41-mer exists in two dimeric forms assigned to the kissing and duplex dimers. The ratio of kissing to duplex form at 5 °C is 0.48 ± 0.22 at I = 113 mM and 0.91 ± 0.35 at I = 183 mM. The observed decrease in K with RNA length suggests that the dimerization constant of the packaging region of HIV-1 is small, <∼105 M-1, implying that the nucleocapsid protein is important in promoting dimerization in the capsid of the virus.
Url:
DOI: 10.1021/bi990744t
Affiliations:
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<wicri:regionArea> Corresponding author: phone (315) 443-4601; Fax (315) 443-4070;e-mail</wicri:regionArea>
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<term>Electrophoresis, Polyacrylamide Gel</term>
<term>HIV-1 (chemistry)</term>
<term>HIV-1 (genetics)</term>
<term>HIV-1 (metabolism)</term>
<term>Humans</term>
<term>Models, Chemical</term>
<term>Molecular Sequence Data</term>
<term>Osmolar Concentration</term>
<term>Peptide Chain Initiation, Translational (genetics)</term>
<term>RNA, Viral (chemistry)</term>
<term>RNA, Viral (genetics)</term>
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<term>Static Electricity</term>
<term>Temperature</term>
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<term>ARN viral (métabolisme)</term>
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<term>Concentration osmolaire</term>
<term>Dimérisation</term>
<term>Données de séquences moléculaires</term>
<term>Humains</term>
<term>Initiation de la traduction (génétique)</term>
<term>Modèles chimiques</term>
<term>Séquence nucléotidique</term>
<term>Température</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1) ()</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (génétique)</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (métabolisme)</term>
<term>Électricité statique</term>
<term>Électrophorèse sur gel de polyacrylamide</term>
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<term>Dimerization</term>
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<term>Humans</term>
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<term>Temperature</term>
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<front><div type="abstract">The genome of the human immunodeficiency virus (HIV) exists as a dimer of two identical RNA molecules hydrogen bonded to each other near their 5‘ ends. The dimer, known to be important for viral infectivity, is formed by two monomers interacting through a stem−loop structure called the dimer initiation site (DIS). An initially formed intermediate, the “kissing” dimer, is unstable and rearranges to the stable, duplex form. In this report we use nondenaturing polyacrylamide gel electrophoresis to measure the monomer−dimer equilibrium constant of three RNA sequences, 41-, 27-, and 19-mers, located in the DIS of the MAL isolate of HIV-1. Experiments in which the RNA was equilibrated at various temperatures before electrophoresis revealed that interconversion is rapid for all the sequences, so that they reach equilibrium in the loading well of the gel at 5 °C before they enter the gel proper. However, interconversion kinetics in the gel are slow, so autoradiographic spot intensities can be used to measure the amounts of monomer and dimer present when the sample entered the gel. After correction for the amount of RNA added with the radiolabel and dilution of samples in the loading well of the gel, dimerization equilibrium constants were calculated from spot intensities. The calculated values of the dimerization constant K at 5 °C were ∼105, ∼106, and ∼108 M-1 for the 41-, 27-, and 19-mers, respectively, in solutions of ionic strength, I, of about 100 mM. The decrease in K by three orders of magnitude between the 19-mer and 41-mer is due in part to the change in rotational entropy of rodlike molecules on dimerization and in part to the increased conformational entropy of the monomers. As expected, increased ionic strength increases the dimerization constant for all three RNAs. For the 41-mer, however, K has a maximum value at I ∼140 mM. The origin of the decrease in K for higher I is unknown but it may be due to formation of species (perhaps higher order oligomers) that do not enter the gel. The 41-mer exists in two dimeric forms assigned to the kissing and duplex dimers. The ratio of kissing to duplex form at 5 °C is 0.48 ± 0.22 at I = 113 mM and 0.91 ± 0.35 at I = 183 mM. The observed decrease in K with RNA length suggests that the dimerization constant of the packaging region of HIV-1 is small, <∼105 M-1, implying that the nucleocapsid protein is important in promoting dimerization in the capsid of the virus.</div>
</front>
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