BIO202_05_Euk_Trxn

Transcription: a primer (eukaryotic)

Aurnab Ghose; 150202

Same genome!

Anise swallowtail, Papilio zelicaon

Same genome!

Gene mRNA Translation at ribosome

Protein

Transcription

Transcription •  Requires

– Transcription initiation site •  where transcription begins

– Promoter •  to which RNA polymerase binds

•  In multicellular eukaryotes – RNA polymerase binds to promoter (TATA

box)

Eukaryotic promoter elements. A eukaryotic promoter usually contains a TATA box located 25-35 bp upstream from the transcription initiation site. One or more UPEs (upstream promoter elements) is usually present.

TATA box Transcription initiation

site UPE TATA A

pre-mRNA

A A

T T

Enhancers

•  Located far away from promoter –  control some gene expression

•  Help form active transcription initiation complex

•  Specific regulatory proteins –  bind to enhancer elements –  activate transcription by interacting with

proteins bound to promoters

Enhancers

actcaaaaaaaaaacggttgggttgcgccatacatatgaaagagtatagaataatgatgtatttcccaaatcaaatatcatggt

aaaatttaaCAATgacccattcggattcattgataatattagttgatggatcatttgtaaaaaggttttattaactcctaagt

tatgtcgagtagaccttgttgttgttgcTATAATTcttaatcATGcgttgtagggggagatttatgtcaccacaaacagaaacgaaagcaaaggttgggttcaaagctggtgttaaagactgaacgtagcagctacgatcgatcgactagctgcatcgggctagcgaagcttcgatcgatcgatcgagctagcgagcccccagttttaggtcgagctttcagctcagctaggcgcgaaatctcgagcgcagctcactagctgctctagcatcgagctacgatcgcgatcgagctagctagaattatccgtgaagcttgcaaatggagtcctgaattagctgctgcttgtgaagtctggaaggaaatcaaatttga

attcccagcaatggatactttgTAAtccagtaataatcattcgttctattaatttccattaaactcggcccaatctt

Transcription •  Is  the  process  of  making  messenger  RNA  (mRNA)  from  a  DNA  template  

•  RNA  polymerase  •  Very  similar  to  DNA  replica;on  •  Remember:  as  in  replica;on,  in  transcrip;on,  addi0on  of  a  new  nucleo0de  occurs  at  the  3’  end!  

•  Transcrip;on  occurs  by  base  pairing:  – A-­‐U:  2  H  bonds;  G-­‐C:  3  H  bonds  

Three Steps of transcription

Complementary Base-pairing of DNA to mRNA

•  If  the  template  strand  reads:      T-­‐A-­‐C-­‐C-­‐T-­‐T-­‐A-­‐A-­‐C-­‐C-­‐G-­‐G-­‐T-­‐T-­‐A    

•   The  transcribed  mRNA  is:      A-­‐U-­‐G-­‐G-­‐A-­‐A-­‐U-­‐U-­‐G-­‐G-­‐C-­‐C-­‐A-­‐A-­‐U  

Transcrip;on  

The structure of mRNA

1.  5’  Guanine  cap  (G-­‐cap)  2.  Leader  sequence:  does  not  get  made  into  protein  3.  Protein  coding  region  begins  with  AUG  &  ends  with  a  STOP  

codon  4.  Trailing  sequence  does  not  get  made  into  protein.  5.  3’  poly-­‐A  tail  

1.  5’ Guanine cap (G-cap) 2.  Leader sequence: does not get made into protein 3.  Protein coding region begins with AUG & ends with

a STOP codon 4.  Trailing sequence does not get made into protein. 5.  3’ poly-A tail

Gene Expression: Translation •  Central  Dogma:    DNA    →      RNA    →      Protein  

– Proposed  by  Francis  Crick  

•  DNA:  3'  ACC  AAA  CCG  AGT  •  mRNA:  5'  UGG  UUU  GGC  UCA    •  Protein:        Trp            Phe      Gly      Ser  

•  The  string  of  amino  acids  has  a  direct  rela;onship  to  nucleo;de  bases  in  RNA  and  DNA  

•  Every  three  nucleo;des  is  called  a  Codon  •  Each  Codon  corresponds  to  ONE  amino  acid  

•  Amino  acid  subunits  are  added  at  the  carboxyl  terminus  of  the  growing  protein  

The elements of eukaryotic transcription

•  Cis-acting elements – Basal promoter elements – UPEs – Enhancers

•  Trans-acting factors – RNA polymerase – Transcription factors – Accessory proteins

Modulating eukaryotic transcription

Promoter proximal elements (UPEs) Enhancer elements: Can function in either orientation.

Can occur far (<50 kb) from the gene. Can be up- or downstream.

Three RNA polymerases in eukaryotes"

I: pre-rRNA"II: mRNA"III: tRNAs, 5S rRNA, " small stable RNAs""!Also differentiated by sensitivity to α-amanitin.!

Complexity of eukaryotic RNA polymerases"

Function of most subunits is unclear, but nearly all the subunits

are essential

Transcription initiation at eukaryotic protein-coding genes

# of subunits Activity

RNA pol II 12 RNA synthesis

General TX factorsTFIID 9 Promoter recognitionTFIIB 1TFIIE 2TFIIF 2TFIIH 9 helicase

Mediator 20

TBP binds to TATA-box DNA"

TATA-binding protein = TBP!!

TBP is a subunit of TFIID!

Pol II won’t bind unless TFs bind first.

Stepwise assembly of Pol II transcription-initiation complex"

Transcriptional control requires still more proteins

RNA pol II (12 proteins) General transcription factors (23 proteins) chromatin remodeling complex (15 proteins) specific transcription factors (? proteins) Mediator complex (20 proteins)