By Alberts

TBP

TATA

By Alberts
By Alberts
Mechanisms of transcription by Pol II 
Structure of RNA polymerase II. Cutaway view, to reveal contents of active center cleft. Surface
representation of atomic model, with features colored as follows: clamp, orange; wall, blue; bridge helix, green;
active center Mg ion, pink; and remainder of polymerase, gray. A) Transcribing complex, with coding strand of
DNA in active center region in turquoise, and RNA in red (PDB 1I6H). B) RNA polymerase II – TFIIB complex,
with backbone model of TFIIB in yellow (PDB 1R5U).
By Kornberg, (2005) FEBS Letters, 579, 899-903
Structure of RNAPII and interaction of the enzyme with promoter DNA. This schematic representation
of the polymerase (shown in orange) emphasizes the way in which the clamp and wall domains restrict access
to the active site. Subunits Rpb4 and Rpb7 form a complex (shown in blue) that can dissociate from the core
enzyme, and might play a role in helping to determine the position of the clamp domain. The Rpb4–Rpb7 complex
may also be involved in interaction with newly synthesized RNA. The narrow configuration of the active site cleft
probably requires melting of the transcription start region for the template strand to reach the RNAPII active site
(indicated by the red dot).

Asturias FJ. 2004. Curr Opin Struct Biol. 14(2):121-9. Review.

RNA polymerase II transcription initiation complex. X-ray and electron microscope
structures (upper left) were assembled in a complete transcription initiation complex
(lower right).
By Kornberg, (2005) FEBS Letters, 579, 899-903
Action of Mediator
 Tail

The yeast RNA Polymerase II holoenzyme revealed by electron microscopy and image processing.
(A) The extended Mediator contains three distinguishable regions; head (h), middle (m),
and tail (t). The globular density embraced by Mediator is identified as RNA polymerase II. The outline of a
projection of the previously determined polymerase three-dimensional structure is superimposed (dark line),
with the point of attachment of the C-terminal domain (dark circle) and the location of the DNA-binding channel
(c) indicated. (B) Tentative subunit organization for the holoenzyme. The model is based on available structural
Information and reported physical interactions. The surface of each subunit has been calculated by assuming
a globular shape and drawn in scale. Subunits in red have reported homologs in Saccharomyces pombe and,
with the exception of Rox3 and Srb6, also in mammalian Mediator. The yellow subunits are specific for
Saccharomyces cerevisiae.
By Bjorklund & Gustafsson, (2004) Advance in Protein Chem., 67, 43-65
Mediator and its interaction with the basal transcription machinery. The structure of the RNAPII–Mediator
complex has revealed the way in which RNAPII interacts with the Mediator complex. As shown, upstream promoter
DNA, IIB and TBP are all expected to be located at the interface between polymerase and Mediator. This implies that
RNAPII and Mediator cannot arrive at a promoter as a pre-formed complex, but must be recruited independently.

Asturias FJ. 2004. Curr Opin Struct Biol. 14(2):121-9. Review.

 1. Poised or committed state of the HNF-4 gene

2. Recruitment of CBP, P/CAF, and Brg-1 to the

enhancer region and assembly of the RNA pol-II
holoenzyme at the proximal promoter region.

3. Unidirectional movement of the DNA-protein

complex formed on the HNF-4 enhancer along
the intervening sequences and spreading of
histone hyperacetylation.

4. Formation of a stable enhancer-promoter

complex, hyperacetylation of nucleosomes
located at the promoter, remodeling of the
nucleosome located at the transcription
start site, and release of RNA pol-II from the
promoter.

Model Depicting the Sequential Steps Involved in the Formation of an Active Pre-initiation
Complex on the HNF-4α Regulatory Region.
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.
What is transcription?
Transcription is a process that cell selectively copies a particular 
portion of its genetic information from DNA ­ a gene ­ to RNA.

Gene A Gene B

TRANSCRIPTION TRANSCRIPTION

Transcription  is a process of DNA template­directed RNA 
polymerization, or an ordered linkage of ATP, CTP, UTP and GTP 
through phosphodiester bonds in 5’ to 3’ direction, with the nucleotide 
order determined by complementary base pairing.
Features:
It uses one DNA strand as a template.
It does not need primers.
It synthesizes RNA from 5’ to 3’(unidirectional).
DNA duplex unwinds upstream and rewinds downstream of the transcription “bubble”.
It involves the function of multiple enzymes and proteins.
 Three principal types of RNA polymerases in human 
cells synthesize RNAs with different functions

Polymerase   Product         Regulation Stability Site of synthesis

I           rRNA         simple    high       nucleolus

II      precursor of mRNA       very diverse     low     nucleoplasm

III      tRNA, small RNA         simple     high            nucleoplasm

Types of RNA Function
mRNAs code for proteins
rRNAs form the structure of ribosomes and catalyze protein synthesis
tRNAs functions as adaptors between mRNAs and amino acids
snRNAs functions in processes such as pre­mRNA splicing

Lectures are focused on RNA polymerase II (Pol II)­mediated 
transcription.
The three stages of transcription:
Initiation, elongation and termination.

Signals that control RNA polymerases where to start 
and stop are encoded in DNA.

Gene A
Gene B

TRANSCRIPTION
TRANSCRIPTION

Initiation is the main point at which cells regulate transcription.
Initiation
The selection of a DNA segment to be transcribed is made by the 
formation of an initiation complex at the promoter.
Promoter: a short region of DNA sequences that specifies the 
association of RNA polymerase complex to initiate transcription.

Consensus sequences of E. coli promoter

5’………TATTGACA………………TATAAT……Start site
        ­35          ­10           +1
The sigma factor in E. coli interacts with the –35 and –10 
sequences at the promoters to select genes for the RNA 
polymerase to transcribe.