Functional Characterisation of the Native NH2- Terminal Transactivation Domain of the Human Androgen Receptor

Binding Kinetics for Interactions with TFIIF and SRC-1a

Derek N. Lavery, Iain J. McEwan

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.
Original languageEnglish
Pages (from-to)3352-3359
Number of pages8
JournalBiochemistry
Volume47
Issue number11
Early online date20 Feb 2008
DOIs
Publication statusPublished - 18 Mar 2008

Keywords

  • RNA-polymerase-II
  • transcription factor IIF
  • carboxyl-terminal domain
  • general transcription
  • dependent transcription
  • phosphatase FCP1
  • RAP74 subunit
  • in-vitro
  • protein
  • coactivator

Cite this

@article{d187fb8c816848c58f7505a7e03a22cb,
title = "Functional Characterisation of the Native NH2- Terminal Transactivation Domain of the Human Androgen Receptor: Binding Kinetics for Interactions with TFIIF and SRC-1a",
abstract = "The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.",
keywords = "RNA-polymerase-II, transcription factor IIF, carboxyl-terminal domain, general transcription, dependent transcription, phosphatase FCP1, RAP74 subunit, in-vitro, protein, coactivator",
author = "Lavery, {Derek N.} and McEwan, {Iain J.}",
year = "2008",
month = "3",
day = "18",
doi = "10.1021/bi702220p",
language = "English",
volume = "47",
pages = "3352--3359",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "11",

}

TY - JOUR

T1 - Functional Characterisation of the Native NH2- Terminal Transactivation Domain of the Human Androgen Receptor

T2 - Binding Kinetics for Interactions with TFIIF and SRC-1a

AU - Lavery, Derek N.

AU - McEwan, Iain J.

PY - 2008/3/18

Y1 - 2008/3/18

N2 - The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.

AB - The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.

KW - RNA-polymerase-II

KW - transcription factor IIF

KW - carboxyl-terminal domain

KW - general transcription

KW - dependent transcription

KW - phosphatase FCP1

KW - RAP74 subunit

KW - in-vitro

KW - protein

KW - coactivator

U2 - 10.1021/bi702220p

DO - 10.1021/bi702220p

M3 - Article

VL - 47

SP - 3352

EP - 3359

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 11

ER -