Gene-L9-DNA transcription I General overview of Eukaryotic transcription, RNA polymerases and key regulatory sequences

what is the genome, transcriptome and proteome?

• GENOME – the total of genetic
  information of the organism as
  embodied in its complete DNA
  sequence

• TRANSCRIPTOME – a full range of RNA
  molecules expressed by an organism
  (protein coding and non-coding)

• PROTEOME – complete set of proteins
  expressed by the organism

what is the definition of transcription? what does it control? what is transcription a response to?

1. synthesis of RNA from DNA template by an RNA polymerase and associated factors

2. controls the protein content of the cell

3. response to a changing cellular environment- involves regulation by transcription factors: immune response, nutrient availability, development and differentiation

similarities(4) vs differences(4) of transcription and replication?

similarities:

1. Enzymes extending a polymer use monomers and ATP

2. Initiated by large protein complex, multiprotein

3. Template directed synthesis - DNA

4. Extension of the chain always goes 5’ to 3’

differences:

1. RNA rather than DNA monomer

2. U-A bps and not T-A

3. There is no need for primers for transcription so no need for primase, the enzyme

4. Only specific regions of genome are transcribed and in replication is the entire thing

what are the 3 steps of transcription?

1. initiation- recruiment of RNA polymerase to the gene promotor

2. elongation- polymerisation of RNA

3. termination- dissociation of RNA polymerase from the DNA template

what main aspects of transcription are commonly regulated? (3- for 3- name 5)

1. access to DNA- chromatin remodelling and histone modification (acetylation opens DNA, methylation and deacetylation close it)

2. activation of TF- usually inactive in the cytosol, needs to be activated by signals, dimerise and move to the nucleus and bind to DNA

3. rate of initiation:

• TF binding- more activators- higher iniation

• promotor strength- strong prmotors- recruit RNA pol more efficientyly

• protein-protein interactions- stabilise initiation complex

• chromatin state- open means easier iniation

• enhancers- increase local conc of transcription machinery

what is the common theme in transcription regulation? and what is required for initiation? DNA, what positions RNA, what else binds?

common theme: recognition of specific DNA binding sites

initiation

• DNA must be accessible to the large multi protein complexes

• TATA box helps position RNA pol correctly

• histone state matters: deacetylated- closed chromatin etc

• activator proteins bind enhancers to increase transcription

• histone removal/remodelling allows TF binding

• TFs act as a docking site for RNA pol

how does the transcription initiation complex form and function? (6)

• RNA polymerase binds to the promotor- TATA box

• general transcription factors assemble too

• additional proteins: chromatin remodelling complexes and HDACs(deacetylases).

• mediator complex links activators to RNA pol

• enhancers bind: can be thousands of nucleotides away- brought by 3d DNA looping

• once assembled, transcription begins

structural and functional features of RNA polymerase during transcription? whats the template

• large, multiprotein complex

• DNA is locally unwound- helicase activity from the TF

• uses ssDNA template to synthesise RNA

• has tunnel/channel where nucleotides enter NTPs

• RNA synthesised- in active site and elongates

• small region of DNA is unwound at a time

3 eukaryotic RNA polymerases and functions

1. RNA pol I- produces ribosomal RNA 18s 5.8S and 28S- nucleolus

2. RNA pol II- mRNA, also miRNA and non coding RNA- located in the nucleoplasm. has CTD and phosphorylation regulates initiation

3. RNA pol III- makes tRNA and 5S rRNA- mainly in the nucleoplasm but also in cytosol for viral response

what makes RNA pol II unique?

has a CTD- carboxyl terminal domain

when it is phosphorylated it regulates initiation- 3 amino acids get phosphorylated and important for initiation

how does eukaryotic RNA pol differ in promotor recognition? what experimental shows what inhibits RNA pol?

• different RNA pol transcribe different types of RNA as they recognise different promotor sequences

• experimental: alpha-amantin a fungal toxin inhibits RNA pol II but not Pol III and I

• proteins separated by increasing salt concentration and RNA pol activity measured- 3 peaks- 1, 2 and 3- with toxin pol II disappears

which DNA strand is used in transcription, what types of RNA are produced in the cell (percentages and what Pol transcribes them)

• transcription strand- antienne strand which is the template strand of DNA

• transcribed 5’→3’

• rRNA: 80%- ribosomal and made by RNA Pol I and III

• tRNA and snRNA 15%- translation and RNA processing- RNA pol I, II, III

• mRNA-5%- prooien coding by RNA pol II

what are the key structural and regulatory features of a typical human gene?

structure:

• regulatory DNA sequence to control transcription

• TSS- transcription start site

• introns/exons- introns removed

regulatory:

1. promotor- near trasncription start site- can be 100bp away and has a TATA box. binds general TF and RNA pol

2. enhancers- can be far from the gene and acts in cis(same DNA molecule). binds regulatory activators and increase transcription

what are the essential elements of eukaryotic core promotors. what is a core promotors? (5)

core promotor: minimal DNA sequence required for transcription initiation

• initiator sequence -3 to +5- includes +1 transcription site. has a CA motif

• TATA box- -25bp. AT rich as less hydrogen bonds to unwind. surrounded by GC- helps RNA pol bind

• DPE- downstream promotor element- downstream transcription start site- can function instead of a TATA box

• CAAT box- upstream- binds TF and increase transcription

• GC box- upstream- binds Sp1

what are enhancers? how do they regulate transcription?

enhancers: DNA sequences that increase transcription levels. can act far away from the gene, 3000bp from the TSS. position is flexible

• binds specific TF and work via DNA looping to interact with promotor

• can increase transcription 100 fold

• acts in cis

• discovered in viruses and found in Ig heavy chain