16. RNA II Promoters

Repressor (trans-acting factor) will block transcription initiation when

bound to the operator (cis-acting element) - negative control

sites for RNAp (promoter) and repressor (operator) could overlap

repressor binding blocks RNAP from interacting with DNA at the start site

affinity of repressor for operator depends on presence/absence of effector

its binding changes repressor’s conformation → change in activity

activators (trans-acting factor) that could

increase binding of RNAp to promoter - positive control

binding sites for activators are called

enhancers (elements)

affinity of activator for enhancer also sometimes depends on

presence/absence of effectr

some proteins are regulators

could execute positive or negative regulation depending on presence/absence of effector → different conformation = affinity for different cis elements

DNA bending (as a consequence of binding of a trans factor) could lead to

negative or positive regulation - regulatory protein can directly contact RNAP - preventing or helping RNAP to interact with DNA at the start site (promoter)

direct contact of RNAp and activator protein causes

conformational changes in RNAP which promote formation of open complex

early studies rRNA genes in eukaryotes

rRNA genes have

1. high GC content

2. repetitive

3. found in nucleolus

in eukaryotes RNA synthesis under various conditions

1. high ionic concentration - RNA with low GC content

2. low ionic concentration - RNA with high GC content

3. Mg2+ low ionic strength - most of transcription in nucleolus

4. Mn2+ high ionic strength - transcription thoruhgout the nucleus

conclusion: more than one RNAP

one could work in nucleolus, sitmulated by low salts and Mg2+, and one would work in nucleoplasm, stimulted by high slats and Mn2+

RNAP I

active at low ionic strength, works with both Mg2+ and Mn 2+

RNAP II

more active at high ionic strength, works better with Mn 2+

RNAP III

active over a broad range of ionic strengths, works better with Mn 2+

alpha-amanitin

RNAPII most sensitive

actinomycin D

RNAP I most sensitive

each one of three RNAPs is a complex enzyme

• 8-14 subunits depending on the RNAP

• 5 subunits are common to all three RNAPs (rpb 5,6,8,10 and 12)

• large subunits similar to B and B’ of Ecoli RNAP

completely different binding sites = promoters

what forms the main 25 A channel in RNAP II and what does it hold

formed by Rbp1 and Rbp2; accommodates ~ 20 bp of double stranded DNA

function of jaws in RNAP II

grads dsDNA at the channel opening to help position it for transcription

which RNAP II components make up the sliding clamp

Rbp 1,2 and 6; keeps polymerase attached to DNA during elongation

RNAP II active site

on Rbp2; contains Mg2_ and a conserved aspartate motif for catalyzing RNA synthesis

Rbp 1 in RNAP II

• Function: Binds DNA and is responsible for α-amanitin sensitivity.

• Has a carboxy-terminal domain (CTD) that can be phosphorylated.

• Two forms:

  • IIα = non-phosphorylated form → binds to promoter (initiation).

  • IIo = phosphorylated form → active during elongation.

• Functional difference: CTD phosphorylation status determines transcription phase.

Rbp 2 in RNAP II

Contains the polymerization active site (catalyzes RNA synthesis).

Rpb 3 in RNAP II

• Contains a 20 amino acid region similar to bacterial α subunit.

• Functions as part of a dimer in the holoenzyme (two monomers like bacterial RNAP α subunits).

CTD Tail in RNAP II

• stretch of 7 aa that are repeated multiple times on RPB1 subunit

  • 5/7 have -OH this is a HYDROPHILIC, phosphorylation site

unphosphorylated CTD used to

initiate transcription

phosphorylated CTD is present only for

high levels of transcription

CDT tail critical for

methyl cap addition, and polyadenylation; splicing

regulation of transcription in eukarytoic gene expression

1. nothing resembling operons is known in eukaryotes exvept in nematodes

2. each eukaryotic gene has specific control sequences (protein binding sites) - gene transcriptions are controlle individually

3. translation in cytoplasm

4. numerous ways expression controlled

measurements of transcription rates reveal that regulation at level of initation of transcription is wide spread

roles of promoters and other cis-acting elemtns

Cannot distinguish between transcription rate and

degradation rate of mRNA

northern, in situ hybridization and microarrays → information about steady-state transcript levels based on

extracted RNA

transcription rate in eukaryotes depends on

1. availability/strength of promoter

2. presence/absence of regulatory proteins

3. availability/strength of their binding sites (cis elements, boxes) on DNA

Run-on, run-off and reporter gene transcripion

→ mRNA is getting transcribed during the assay

info about the actual efficiency of transcription rate

These assays are used to locate and assay teh promoter region

(presence/absence of all necessary elements, their position and availability)

how reporter gene is used to asses promoter strength

the promoter of interest is cloned upstream of the reporter gene, and the amount of reporter product produced is proportional to the promoter’s strength

5’ deletion series

method where progressive deletions are made from the 5’ end of the promoter to determine which regions are important for its activity

promoter sequences must be located close to the

start point are required for initiation fo transcription

class II promoters have two pars

1. core elements (also called core promoter or only promoter)

2. regulatory elements (one of them is enhancer)

core promoter contains

TATA box ~ 25-35 bp upstream from start site

• similar in action to E.coli TATA box, but further upstream

TATA-less promoters

in housekeeping genes absolutely necessary genes or in specialized genes - produced only in certain cells - these genes must have either initatior (core element) or GC boxes (classified as upstream elements) to start transcription

functions of TATA box

1. locates the start of transcription ~ 30 bp downstream

2. sometimes important for the efficiency of transcription

3. TATA-binding protein (TBP) binds to TATA box and initiates the assembly of general transcription factors and RNA polymerase

GC boxes summary

• orientation independent

• must be close to TATA box, usually present in housekeeping genes

• could be a part of TATA-less promtoer

RESULT: mRNAs with multiple alternative 5’ ends (UTRs)

CCAAT box

• enhancer

• factor is CTF

• no prokaryotic equivalent - must be necessary in eukaryotes

promoter-proximal elements

• control regions 100-200 bp upstream from start site

• cell type specific - specific “set” of elements ditates expression

• identified thorugh 5’ deletion series

insulators from chromatin boundary

between euchromatic and heterochromatic regions in an individual chromosome

loops formed by CTCF and Cohesin access is under the control of

adjacent locus control regions

insulated neighbourhoods

regions of DNA within extruded “loops” of eukaryotic DNA. chromosome territories are respected

Upstream activating sequences

in yeast only

• contain only one UAS

• within 100 bp upstream of promoter

core elements

1. TATA box

2. initatior

3. downstream element

4. TF II B recognition

regulatory elements

1. upstream

   • GC boxes

   • CCAAT

   • promoter proximal elements

2. enhancers and silencers

3. boundary elements and insulators