Transcription of the Acanthamoeba TATA-binding Protein Gene: A SINGLE TRANSCRIPTION FACTOR ACTS BOTH AS AN ACTIVATOR AND A REPRESSOR

Weibiao Huang,Erik Bateman

doi:10.1074/jbc.272.6.3852

Abstract

Transcription of the Acanthamoeba TATA-binding protein (TBP) gene is regulated by TBP promoter-binding factor (TPBF), a previously described transactivator that binds as a tetramer to the TBP Promoter Element (TPE) and stimulates transcription up to 10-fold in vitro. Here we report that TPBF also functions as a transcription repressor by binding to a negative cis-element, located between the TATA box and the transcription initiation site. The negative element, referred to as the nTPE, is structurally similar to the TPE, and its disruption increases the transcription potency of the TBP promoter. TPBF binds to the nTPE, as demonstrated by mobility shift assays. However, the binding affinity of TPBF for the nTPE is about 10-fold lower than for the TPE. When placed upstream of the TATA box, the nTPE has very little effect on transcription. However, it inhibits transcription when placed at several positions downstream of the TATA box. Mechanistic studies with the TBP promoter suggest that binding of TPBF to the nTPE not only prevents TBP from binding to the TATA box but also displaces bound TBP, thereby inhibiting further assembly of the preinitiation complex. These results suggest a mechanism in which the cellular TPBF concentration controls the level of TBP gene transcription and show that a single factor can be stimulatory, inhibitory, or neutral depending on the sequence and the context of its binding site.

Highlights

Transcription of the Acanthamoeba TATA-binding protein (TBP) gene is regulated by TBP promoter-binding factor (TPBF), a previously described transactivator that binds as a tetramer to the TBP Promoter Element (TPE) and stimulates transcription up to 10-fold in vitro
These results suggest a mechanism in which the cellular TPBF concentration controls the level of TBP gene transcription and show that a single factor can be stimulatory, inhibitory, or neutral depending on the sequence and the context of its binding site
The TBP Gene Promoter Contains a Negative cis-Element between the TATA Box and the Transcription Initiation Site—Inspection of the DNA sequence between the TATA box and the transcription initiation site of the TBP gene promoter identifies a sequence that is similar to the upstream TPBF recognition site, the TPE, but inverted (Fig. 1A)

Summary

A SINGLE TRANSCRIPTION FACTOR ACTS BOTH AS AN ACTIVATOR AND A REPRESSOR*

(Received for publication, September 12, 1996, and in revised form, November 12, 1996). Transcription of the Acanthamoeba TATA-binding protein (TBP) gene is regulated by TBP promoter-binding factor (TPBF), a previously described transactivator that binds as a tetramer to the TBP Promoter Element (TPE) and stimulates transcription up to 10-fold in vitro. Mechanistic studies with the TBP promoter suggest that binding of TPBF to the nTPE prevents TBP from binding to the TATA box and displaces bound TBP, thereby inhibiting further assembly of the preinitiation complex These results suggest a mechanism in which the cellular TPBF concentration controls the level of TBP gene transcription and show that a single factor can be stimulatory, inhibitory, or neutral depending on the sequence and the context of its binding site. TATA-Binding Protein (TBP) is a highly conserved eukaryotic basal transcription factor that is required for transcription by all three RNA polymerases both in vitro and in vivo (10 –12). A working model of how TPBF regulates TBP gene expression is presented

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION