Molecular Biology

Chapter 11b Outline

Class II preinitiation complex:

∑ SL1

a) Directs accurate initiation of RNA PolI (F11.39); species specific.

b) Does not cause a footprint by itself (F11.40); when combined with a PolI fraction it causes a footprint in the UCE (sites A and B) and in the core promoter element; enhanced cleavage during a DNase footprint assay.

c) The core promoter element also determines species specificity (F11.41); SL1 is required for PolI transcription; the UCE is independent of species specificity.

∑ UBF

a) Identified in the RNA PolI fraction as the DNA binding protein (F11.42); footprints the UCE (site A) and core element; SL1 enhances UBF binding and extends the UBF footprints in the UCE and core element.

b) UBF is not sufficient for transcription (F11.43); UBF enhances SL1 dependent PolI transcription; if SL1 enhanced UBF binding in site B is blocked, UBF is incapable of stimulating transcription even in the presence of SL1 (F11.44).

∑ TBP

a) Co-purifies with SL1 (F11.45); heparin agarose chromatography; inhibition of SL1 activity in transcription using TBP antibodies (F11.46); TAF_I110, TAF_I63 and TAF_I48 are also found in SL1; SL1 is analogous to TFIID for PolII transcription.

Class III preinitiation complex:

∑ TFIIIA, TFIIB and TFIIIC participate in PolIII transcription; TFIIIA, B and C are required for 5S RNA transcription, but TFIIIA is not required for tRNA transcription (F11.48).

∑ TFIIIA was the first eukaryotic transcription factor to be isolated; it is a zinc finger protein; zinc finger proteins use 4 amino acids (cysteines and/or histidines) to bind zinc; once zinc is bound, the protein can bind to the major groove of the DNA.

∑ Extracts containing TFIIIB, TFIIIC and PolIII protect box B and upstream when transcription is disabled, but also box A when transcription can occur (F11.49); TFIIIC binds to the box A and box B (F11.50); TFIIIB binds tightly to the upstream sequence even after TFIIIC is removed (F11.50); TFIIIB binding requires prior binding of TFIIIC (tRNA promoter) or TFIIIC and TFIIIA (5S RNA promoter) (F11.51); TFIIIC initiates formation of the preinitiation complex (F11.52).

∑ TBP is a component of TFIIIB; TFIIIB also contains 2 TAF_IIIS: TAF172 and TAF-L; in Drosophila, TRFI is required for tRNA and 5S RNA transcription instead of TBP.

∑ Three unifying principles for core transcription complexes (F11.54): (1) preinitiation complex formation begins with an assembly factor binding to a specific site on the promoter, (2) TBP plays and organizing role by brining in additional factors and polymerase, and (3) the specificity of TBP is governed by the TAFs with which it associates.