Quantitative Analysis of the Binding of Turnip Crinkle Virus Coat Protein to RNA Fails to Demonstrate Binding Specificity but Reveals a Highly Cooperative Assembly Interaction

J. M. Skuzeski, Thomas J Morris

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

An element(s) within a 386-nucleotide segment (TCV-386 RNA) of the turnip crinkle virus (TCV) genome was previously implicated in virus assembly nucleation. To localize the proposed high-affinity binding determinants, we analyzed the ability of the coat protein to bind the full-length and truncated derivatives of the TCV-386 RNA using gel retardation and nitrocellulose filter retention assays. Quantitation of the binding data indicated that the coat protein did not preferentially recognize a particular region of the RNA Moreover, the affinity of the coat protein for the TCV-386 RNA [apparent dissociation constant (Kd)≈0.5μM) did not appreciably differ from its affinity for other comparably sized RNAs tested, including nonviral RNAs. However, the quantitative studies also suggested that the coat protein binds RNA in a cooperative manner and this was supported by evidence that all of the RNAs examined were bound by multiple copies of the coat protein Based on the number of binding intermediates which could be detected in titrations involving RNAs of different chain length, it appeared that each coat protein binding unit occupies 35-40 nucleotides, Our results demonstrate that encapsidation of TCV RNA results from highly cooperative binding of the coat protein on the large viral genome. However, we were not able to confirm that assembly is mediated by initiation at a high-affinity binding site on the viral RNA.

Original languageEnglish (US)
Article number71319
Pages (from-to)82-90
Number of pages9
JournalVirology
Volume210
Issue number1
DOIs
StatePublished - Jun 20 1995

Fingerprint

Carmovirus
Capsid Proteins
RNA
Nucleotides
Virus Assembly
Collodion
Viral Genome
Viral RNA
Protein Binding

ASJC Scopus subject areas

  • Virology

Cite this

Quantitative Analysis of the Binding of Turnip Crinkle Virus Coat Protein to RNA Fails to Demonstrate Binding Specificity but Reveals a Highly Cooperative Assembly Interaction. / Skuzeski, J. M.; Morris, Thomas J.

In: Virology, Vol. 210, No. 1, 71319, 20.06.1995, p. 82-90.

Research output: Contribution to journalArticle

@article{d2f22221c51a4d90925b247e38990f2f,
title = "Quantitative Analysis of the Binding of Turnip Crinkle Virus Coat Protein to RNA Fails to Demonstrate Binding Specificity but Reveals a Highly Cooperative Assembly Interaction",
abstract = "An element(s) within a 386-nucleotide segment (TCV-386 RNA) of the turnip crinkle virus (TCV) genome was previously implicated in virus assembly nucleation. To localize the proposed high-affinity binding determinants, we analyzed the ability of the coat protein to bind the full-length and truncated derivatives of the TCV-386 RNA using gel retardation and nitrocellulose filter retention assays. Quantitation of the binding data indicated that the coat protein did not preferentially recognize a particular region of the RNA Moreover, the affinity of the coat protein for the TCV-386 RNA [apparent dissociation constant (Kd)≈0.5μM) did not appreciably differ from its affinity for other comparably sized RNAs tested, including nonviral RNAs. However, the quantitative studies also suggested that the coat protein binds RNA in a cooperative manner and this was supported by evidence that all of the RNAs examined were bound by multiple copies of the coat protein Based on the number of binding intermediates which could be detected in titrations involving RNAs of different chain length, it appeared that each coat protein binding unit occupies 35-40 nucleotides, Our results demonstrate that encapsidation of TCV RNA results from highly cooperative binding of the coat protein on the large viral genome. However, we were not able to confirm that assembly is mediated by initiation at a high-affinity binding site on the viral RNA.",
author = "Skuzeski, {J. M.} and Morris, {Thomas J}",
year = "1995",
month = "6",
day = "20",
doi = "10.1006/viro.1995.1319",
language = "English (US)",
volume = "210",
pages = "82--90",
journal = "Virology",
issn = "0042-6822",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Quantitative Analysis of the Binding of Turnip Crinkle Virus Coat Protein to RNA Fails to Demonstrate Binding Specificity but Reveals a Highly Cooperative Assembly Interaction

AU - Skuzeski, J. M.

AU - Morris, Thomas J

PY - 1995/6/20

Y1 - 1995/6/20

N2 - An element(s) within a 386-nucleotide segment (TCV-386 RNA) of the turnip crinkle virus (TCV) genome was previously implicated in virus assembly nucleation. To localize the proposed high-affinity binding determinants, we analyzed the ability of the coat protein to bind the full-length and truncated derivatives of the TCV-386 RNA using gel retardation and nitrocellulose filter retention assays. Quantitation of the binding data indicated that the coat protein did not preferentially recognize a particular region of the RNA Moreover, the affinity of the coat protein for the TCV-386 RNA [apparent dissociation constant (Kd)≈0.5μM) did not appreciably differ from its affinity for other comparably sized RNAs tested, including nonviral RNAs. However, the quantitative studies also suggested that the coat protein binds RNA in a cooperative manner and this was supported by evidence that all of the RNAs examined were bound by multiple copies of the coat protein Based on the number of binding intermediates which could be detected in titrations involving RNAs of different chain length, it appeared that each coat protein binding unit occupies 35-40 nucleotides, Our results demonstrate that encapsidation of TCV RNA results from highly cooperative binding of the coat protein on the large viral genome. However, we were not able to confirm that assembly is mediated by initiation at a high-affinity binding site on the viral RNA.

AB - An element(s) within a 386-nucleotide segment (TCV-386 RNA) of the turnip crinkle virus (TCV) genome was previously implicated in virus assembly nucleation. To localize the proposed high-affinity binding determinants, we analyzed the ability of the coat protein to bind the full-length and truncated derivatives of the TCV-386 RNA using gel retardation and nitrocellulose filter retention assays. Quantitation of the binding data indicated that the coat protein did not preferentially recognize a particular region of the RNA Moreover, the affinity of the coat protein for the TCV-386 RNA [apparent dissociation constant (Kd)≈0.5μM) did not appreciably differ from its affinity for other comparably sized RNAs tested, including nonviral RNAs. However, the quantitative studies also suggested that the coat protein binds RNA in a cooperative manner and this was supported by evidence that all of the RNAs examined were bound by multiple copies of the coat protein Based on the number of binding intermediates which could be detected in titrations involving RNAs of different chain length, it appeared that each coat protein binding unit occupies 35-40 nucleotides, Our results demonstrate that encapsidation of TCV RNA results from highly cooperative binding of the coat protein on the large viral genome. However, we were not able to confirm that assembly is mediated by initiation at a high-affinity binding site on the viral RNA.

UR - http://www.scopus.com/inward/record.url?scp=0029054746&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029054746&partnerID=8YFLogxK

U2 - 10.1006/viro.1995.1319

DO - 10.1006/viro.1995.1319

M3 - Article

VL - 210

SP - 82

EP - 90

JO - Virology

JF - Virology

SN - 0042-6822

IS - 1

M1 - 71319

ER -