Transcription and its control II

transcription and its control in eukaryotic cells

three different multi-subunit RNA polymerase complexes (I, II, III)

many additional proteins (transcription factors) needed to recognize promoter and initiate transcription

transcription factors

have DNA-binding domains and one or more regulatory domains used to interact with RNA polymerases and other proteins involved in transcription

some are common to all three RNA pol complexes, others are complex specific

RNA polymerase I

transcribes pre-rRNA genes (18S, 5.8S, and 25/28S rRNA but not 5S rRNA) in the nucleolus; such genes are arranged in operons, as in prokaryotes

comprised of 13 subunits plus additional transcription factors, including the important transcription factor TATA binding protein (TBP)
in yeast, ~80% of all transcription is dedicated to pre-rRNA synthesis

apparently little in the way of regulation (rRNAs are ubiquitously expressed)

RNA polymerase III

14 subunits in this complex, transcribes pre-tRNA, 5S rRNA, and other small RNA genes

TATA binding protein (TBP) one of numerous protein factors required for this transcription

promoters are unusual: they can have elements located within the genes whose transcription they promote

this complex lands on top of gene and sometimes makes multiple RNA transcripts before it dissociates

RNA polymerase II

transcribes mRNA genes, microRNAs, and some of the small nuclear RNA genes involved in splicing

12 subunit polymerase complex, with many additional protein factors needed for transcription initiation

initiation involves recognition of the TATA box

promoter recognition by TFIID complex

largest subunit has a long carboxyl-terminal domain (CTD) with heptad amino acid repeats (plays critical role in this complex’s function)

TATA box

cis element, important for transcription initiation

proximal control element recognized by TBP

many but not all Pol II promoters have this, most common core promoter element

~20-30 nt upstream of the start site for transcription

TFIID complex

includes TATA binding protein (TBP) and TBP-associated factors (TAFs)

recognizes the TATA box, recruits TFIIA and TFIIB; has positive and negative regulatory functions

control element

a DNA sequence that can be bound by a transcription factor, can be proximal or distal to gene

enhancer

a control element that can be bound by trans-acting factors and thus influence transcription of a gene but is far removed from that gene’s promoter. can be upstream or downstream of a gene

most eukaryotic genes are regulated by

multiple transcription-control elements and binding factors: very complex in multicellular organisms

RNA polymerase II transcription cycle

1. Pol II recruited to DNA by transcription factors. TFIID, Pol II, and DNA form the pre-initiation complex

2. formation of transcription bubble- CTD domain is present but not yet phosphorylated, so this is still just an initiation complex

3. phosphorylation of CTD during initiation. TFIIB, TFIIE, TFIIH leave the complex and thus the elongation complex forms

4. elongation, then termination complex is formed

5. transcription terminates and CTD is dephosphorylated

cis-acting control elements

located within the DNA sequence

can be proximal (e.g. TATA box) or distal (e.g. enhancer) to promoter

can be general or specific to certain genes/gene families

trans-acting factors

any diffusible element that is not located within the DNA sequence, i.e. proteins

transcription factors can be general (e.g. TBP) or specific (e.g. some TAFs)

transcription factors serve to recruit RNA pol II core machinery (analogous to sigma factors in prokaryotes)

most such factors do not participate in transcription elongation (TFIIF is exception)

CTD domain of largest pol LL subunit

PTSPSYS heptad repeats are essential: phosphorylation of hydroxyl-containing residues necessary for transition from initiation to elongation

eukaryotic transcription termination

in vitro experiments suggest that RNA pol II can terminate at multiple sites well beyond the 3’ end of the coding region

AAUAAA sequence acts as a signal for endonuclease cleavage

poly (A) tail added by polyadenylate (poly A) polymerase which also has endonuclease functionality

CTD domain of RNA pol II makes contacts with various mRNA processing factors

How do enhancers influence transcription from so far away?

proteins bind/interact with both proximal and distal cis elements—possible because of DNA looping

transcription activators bind distal regulatory sequences (e.g. enhancers)

multi-subunit co-activators bind activators and act as bridges between the activators and RNA polymerase

in contract, repressor proteins disrupt/prevent contacts between RNA pol II and activators/co-activators