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Gene  Regulation  
Outline:  
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Gene  expression  -­‐  Central  Dogma  
Why  is  Gene  Regulation  important?  
General  principles  of  Gene  Regulation  
Components  of  Gene  Regulation  
o   RNA  polymerase  
o   cis-­‐acting  elements  
o   trans-­‐acting  elements  

Learning  Objectives:  
Why  is  Gene  Regulation  important?  
-   Gene  expression  is  costly  to  the  cell  
The  differences  between  prokaryotic  and  eukaryotic  gene  regulation  
-   Spatial  and  temporal  
-   Operons  vs  Single  transcriptional  units  
-   mRNA  processing:  splicing  
Different  types  of  gene  regulation  
-   Positive  regulation  
-   Negative  regulation  
What  are  cis  and  trans  elements  
-   CisàDNA  
-   TransàProtein  
How  can  you  identify  cis  and  trans  elements  
-   DNA  footprinting,  reporter  assays,  Gel  shift  assays,  DNA  affinity  chromatography  

 
Transcription  has  happened  and  control  the  translation  of  protein  at  the  mRNA  level  

Why  is  gene  regulation  important?  

 
Very  costly  to  have  all  the  genes  switch  on  all  they  time  (use  up  a  lot  of  energy)  
Gene  regulation  is  the  way  to  switch  genes  on  and  off  

 
Gene  expression  –  where?  
Prokaryote:  No  nucleus  to  separate  processes  of  transcription  &  translation  so  when  bacterial  genes  
are  transcribed  into  mRNA  transcripts  they  can  immediately  be  translated  into  protein  
Transcription  and  translation  are  coupled  in  the  cytoplasm  
Eukaryote:  Transcription  &  translation  are  spatially  &  temporally  separated  i
...
 transcription  occurs  
in  the  nucleus  →  pre-­‐mRNA    
Pre-­‐mRNA  processed  (splicing,  polA  tail,  capping)  →  mature  mRNA  which  exits  the  nucleus  &  is  
translated  in  cytoplasm  where  ribosome  can  now  bind  
Transcription  happens  first  before  translation  
 
 

General  principles  of  Gene  Regulation  

 
On  this  gene  locus:  
Open  reading  frame  –  target  gene  that  we  want  to  transcribe  an  translate  into  protein    
Start  and  stop  codon  –  encoded  in  the  DNA  that  initiate  transcription  and  termination  
Promoter  –  stretch  of  DNA  that  allows  DNA  polymerase  to  bind  
Upstream  or  downstream  or  within  promoter  –  binding  elements  call  regulatory  sequences,  
regulatory  proteins  can  have  positive  or  negative  effects  when  bound  
 
Gene  Architecture  in  Prokaryotes:  Operons  
One  promoter  that  drives  several  genes  at  the  same  time,  a  very  long  mRNA  that  encodes  4-­‐5  
different  proteins    
RNA  polymerase  can  bind  to  the  
black  sites  and  transcribe  individual  
gene  (A,  B,  C,  D)  
They  are  all  separate  entities  with  
start  and  stop  codons  within  one  set  
Hence,  they  are  polycistronic  
This  is  because  translation  is  not  
coupled  to  transcription  
•  

Genes  are  contiguous  
segments  of  DNA  that  are  co-­‐linear  with  the  mRNA    

•  

mRNAs  are  often  poly-­‐cistronic  

 
 
 
 

Gene  Architecture  in  Eukaryotes:  Single  transcriptional  units  
Eukaryotes  –  every  single  gene  will  have  its  own  promoter  (monocistronic)  

 
•  

Coding  sequences  are  often  interrupted  by  intervening  sequences  (introns)  

Alternative  splicing  can  produce  multiple  proteins  from  the  same  mRNA  by  different  order  of  
combinations  of  exons  
General  principles  of  Gene  Regulation  

 
RNA  polymerase  can  bind  to  the  promoter  of  a  gene,  but  the  binding  is  not  strong  for  the  initiation  
to  occur  to  make  the  mRNA  
Therefore,  we  need  an  activator,  it  doesn’t  bind  to  the  regulatory  sequence  until  it  gets  activated  by  
a  signal  in  the  form  of  molecules/light/phosphorylation  etc  
Signal  binds  and  induce  a  conformational  change  which  usually  is  a  dimerization  of  the  regulatory  
proteins  binding  together,  then  bind  to  the  DNA  and  influence  the  strength  of  the  binding  of  the  
polymerase
...
 Polymerase  gets  
released  and  start  transcription
...
 The  area  which  is  covered  by  the  protein  is  where  the  regulatory  sequence  is  
and  cannot  be  chewed  up
...
   
 
 
 
 

How  to  identify  cis  elements  
Reporter  Gene  Assay:  Promoter  mapping  

 
Take  the  regulatory  unit  of  the  DNA  and  with  PCR  make  different  size  versions  of  the  regulatory  DNA  
Primer  1+4  gives  the  entire  section  of  the  regulatory  unit,  cis  regulatory  unit  in  green  and  promoter  
region  in  blue
...
 If  negatively  regulated  promoter,  we  don’t  get  green  fluorescence,  but  this  is  not  100%  
negative  as  the  repressor  protein  may  sometime  falls  off  (equilibrium  between  the  bound  and  
unbound  state)  and  the  polymerase  can  quickly  bind  to  promoter  and  transcribe
...
 Therefore,  only  little  transcription
...
 
By  producing  lots  of  cunctation,  we  slowly  reduce  the  length  of  the  regulatory  parts  of  the  gene  and  
observe  the  outcomes  to  map  where  these  regulatory  units  are
...
   
Then  using  low  salt  wash  to  clear  up  unwanted  substances  that  stick  to  pieces  of  DNA  (ie  positively  
charge)  
Now  repeat  the  procedure  but  matrix  only  contains  DNA  sequence  that  the  protein  of  interest  would  
bind  to
...
 
 
 
 

  
Working  out  whether  protein  will  bind  to  DNA  or  not  
Run  a  PAGE-­‐gel  without  SDS  (protein  in  its  native  form),  load  on  a  sample  with  just  DNA  on  the  first  
column
...
 If  protein  binds  onto  the  DNA,  there  will  be  a  shift  
in  the  gel  because  the  protein  will  retard  the  DNA’s  speed  through  the  gel
...
 3rd  column,  is  accessory  protein  binds  to  the  already  bound  
protein  on  the  DNA
...
 
(Normally  use  an  antibody  to  check  for  supershift  and  protein’s  identity)  
Learning  Objectives  
What  makes  up  an  operon  
-   Promoter,  Operators,  structural  genes  
Structure  and  function  of  RNAP  
-   Apo  RNAP  Subunits:  β,  β’,  αi,  αii,  ω    
-   Holo  RNAP  additional  subunit:  σ  
-   Closed  compex  à  Open  complex  
Lac  Operon  
-   Negative  regulation  
-   Activation  through  catabolite  repression  
Arabinose  Operon  
-   Negative  and  positive  regulation  through  AraC  binding  
-   Activation  through  catabolite  repression  
-   Autoregulation  of  AraC  
Tryptophan  Operon  
-   Negative  regulation  

-   Additional  negative  regulation  through  attenuation  
Operon  architecture  

 
•  

Prokaryotic  cells  have  linear  sequences  of  DNA  called  operons    

•  

 An  operon  is  composed  of  a  promoter  sequence,  followed  by  an  operator,  followed  by  one  
or  more  structural  genes,  functional  genes  that  need  to  be  controlled(blueprints  for  
proteins)  

•  

 Operons  are  controlled  by  regulatory  genes  found  elsewhere  on  the  chromosome  which  
regulates  the  expression  of  the  structural  genes  in  response  to  an  environmental  signal  

•  

This  regulation  can  be  up  or  down  regulate  

•  

Ribosome  binding  site  (black  boxes)  

Prokaryotic  RNA  polymerase  

 
Beta  and  beta’  can  associate  with  any  sequences  on  the  DNA  physically  
Alpha  1  and  2  recognise  upstream  promoter  elements  and  interact  with  the  betas
...
 Transcription  factor  binds  to  the  
cis  regulatory  element,  up  regulate  RNAP,  through  alpha  1&2
...
   

 
Different  types  of  sigma  units:  
Initiation  of  transcription  by  the  RNAP-­‐σ70  (A)  and  RNAP-­‐σ54  (B)  holoenzymes
...
 In  contrast,  the  σ54  factor  directs  the  binding  of  RNAP  to  conserved  −12  (TGC)  
and  −24  (GG)  promoter  elements  that  are  part  of  the  wider  consensus  sequence  
YTGGCACGrNNNTTGCW  (where  uppercase  type  indicates  highly  conserved  residues,  lowercase  type  
indicates  weakly  conserved  residues,  N  is  nonconserved,  Y  is  pyrimidines,  R  is  purines,  and  W  is  A  or  
T)  (10)
...
 In  order  to  form  
the  transcription  “bubble,”  a  specialized  activator  (a  bacterial  enhancer  binding  protein  [bEBP])  must  
bind  and  use  the  energy  from  ATP  hydrolysis  to  remodel  the  holoenzyme
...
 In  the  absent  of  lactose,  it  will  bind  to  the  cis  regulatory  unit  –  operator,  just  upstream  of  
the  promoter
...
 (No  need  to  metabolise  
lactose  when  they  are  not  present)  
When  lactose  is  imported  into  the  cell,  it  is  converted  into  allolactose  which  is  the  inducer  molecule  
that  binds  to  the  lac  repressor  protein  and  stops  it  from  binding  to  the  regulatory  element  (DNA)
...
 High  level  of  glucose  
ends  up  inhibiting  adenylate  cyclase,  so  no  cAMP  being  produced
...
 The  complex  then  binds  to  the  regulatory  sequence  upstream  of  the  promoter  and  
interact  with  the  alpha  domains  of  the  RNAP
...
 
 
 

•   Negative  regulation  via  LacI  repressor  
•  

Lactose  binds  to  and  inactivates  LacI  

•  

Catabolite  repression  via  glucose  

•  

Positive  regulation  via  CAP/cAMP  

 
 
 
The  arabinose  operon  
AraB  ,  AraA  and  AraD  are  required  for  the  breakdown  of  arabinose  

 
If  glucose  and  lactose  aren’t  present,  we  can  use  the  linear  sugar  arabinose  (carbon  source)  
AraBAD  are  the  structural  genes  
Arac  is  the  trans  regulatory  protein  that  encodes  the  regulatory  protein  AraC  
AraO2,  araO1  and  aral  are  the  regulatory  regions  and  a  catabolite  binding  site  CAP  cAMP  

 

In  the  absent  of  arabinose,  AraC  binds  to  two  sites,  araO2  and  aral1
...
 There  is  
therefore  no  space  for  the  RNAP  to  bind
...
 Instead  of  binding  to  I2  and  O2,  it  now  binds  to  I1  and  I2
...
 Simultaneously,  the  adenylate  cyclase  is  activated  in  a  low  glucose  level  to  form  
CAP  cAMp  that  binds  to  the  catabolite  site
...
 
Alter  regulation,  araC  acts  as  both  negative  and  positive  regulator
...
   

 
Self-­‐regulation:  AraC  made  can  binds  to  the  promoter  that  drives  araC  and  repress  the  production  of  
araC
...
 Then  level  of  araC  in  the  
system  falls
...
 Always  at  a  constant  level!  
When  no  araC  proteins  are  present,  RNAP  is  free  to  transcribe  the  gene  that  codes  for  the  
production  of  araC  proteins
...
 This  physically  
prevents  transcription  of  the  araC  gene
...
 During  
repression,  a  dimeric  form  of  the  araC  protein  binds  I1  and  O2  site  forming  a  DNA  loop
...
 DNA  returns  to  its  
linear  shape  and  RNAP  can  bind
...
 Trp  recognises  the  protein  and  will  bind  or  
dissociate  with  the  repressor  depending  on  the  concentration
...
 (Lac  repressor  
protein  always  bind  to  the  DNA  and  require  the  present  of  allolactose  for  it  to  fall  of  the  DNA
...
 This  gives  a  70-­‐fold  repression  between  on  and  off
...
 At  the  beginning  of  the  structural  
gene,  there  is  an  attenuated  sequence  gets  transcribed  as  a  single  piece  of  mRNA
...
       

  
How  attenuation  works?  
Two  processes  are  taking  place  at  the  same  time,  both  transcription  and  translation
...
 In  part  1,  there  are  two  codons  that  encode  for  tryptophan
...
 Riboseme  stalls  and  the  mRNA  sequence  at  position  2  and  3  recognise  each  other  and  form  a  
stem-­‐loop
...
 Structural  genes  are  being  transcribed
...
 Ribosome  encounters  
the  Trp  codon,  but  as  there  are  lots  of  Trp  ready  to  be  use,  so  it  just  rushes  through  and  doesn’t  stall  
at  position  1
...
 Sequence  3  binds  to  
sequence  4  instead  and  makes  a  transcriptional  terminator
...
 RNAP  does  not  transcribe  structural  genes
...
 Only  happen  in  prokaryotes!  

Learning  Objectives  
Chromatin  remodelling  
-   CpG  Islands,  methylation/acetylation,  Histone  binding  
Structure  and  function  of  Eukaryotic  RNAP  
-   RNAPI/II/III    
-   TBP,  TFIIB,  TFIIE,  TFIIH,  RNAPII  bind  core  promoter  
-   Many  additional  transcription  factors  
mRNA  splicing  
-   Binding  of  snRNP  to  splice  junctions  
-   Excision  of  intron  sequences  
Gal  Pathway  
-   Activation  of  expression  through  GAL4  
-   Repression  of  GAL4  activity  by  GAL80  
-   Repression  of  GAL80  repression  by  GAL3  and  galactose  
miRNA  gene  regulation  
-   RNA:RNA  interactions  act  as  post  transcriptional  control  of  protein  production  
 

 

Everything  is  always  switch  off  in  
eukaryotes  until  there  is  a  signal  to  turn  a  
gene  on
...
 When  histones  are  methylated,  
they  stay  positively  charged
...
 Then  promoter  becomes  
available
...
   
Acetylation  is  done  by  acetyl  transferase,  
transferring  an  acetyl  group  from  acetyl  
CoA  onto  the  histones
...
 Upstream  regions  that  are  called  CpG  islands  (Cytosine  poly  
Guanine  Island),  this  sequence  of  DNA  can  be  methylated
...
 When  demethylated,  cis  acting  elements  can  now  reopen  
for  trans  regulatory  proteins
...
 
•   Many  genes  in  human  genome  have  upstream  CG-­‐rich  regions  (CpG  islands)  
•   Different  cell  types  have  different  methylation  patterns  (different  gene  expression)  
DNA  is  about  2-­‐3m  long,  but  only  up  to  about  30cm  of  the  DNA  is  used  to  transcribe  the  20000  genes  
our  bodies  use
...
   
Differentiation  between  different  cells  come  from  this,  when  cells  are  exposed  to  different  
environments,  they  cause  our  DNA  to  condense  in  different  ways,  so  different  parts  of  the  
chromosome  are  exposed
...
       

 
DNA  is  methylated,  so  no  transcription  takes  place,  so  DNA  does  not  have  to  be  in  a  relaxed  form
...
 
Histone  becomes  positively  charged  and  DNA  associate  with  the  histones
...
8s,  18s  
and  28s  rRNA  
Small  ribosomal  RNA  that  are  part  of  the  
ribosome  structure  that  are  responsible  for  
recognising  specifics  sequences  on  the  
ribosome  binding  site  
 
 
 
 
RNAP  III  is  responsible  for  5s  rRNA,  all  the  
tRNAs  and  snRNA  
tRNAs:    recognises  codons  and  charge  with  
an  amino  acid  to  build  polypeptide  chain  
snRNA:  small  RNA  that  recognises  splice  
junctions  
 
 
 
General  transcription  
factors  have  to  bind  in  
order  to  initiate  
transcription  
Pol  II  is  made  up  of  12  
domains,  additional  
transcription  factors  have  
to  bind  DNA  in  a  hierarchal  
order  for  polymerase  to  
bind    
All  initiation  transcription  
factors  work  in  concert  doing  the  same  job  as  the  sigma  subunit  in  prokaryotes  (recognition  of  the  
promoter  sequences  for  POL  to  bind)  
On  top  of  that,  there  are  transcription  factors  (activators  or  repressors)  that  bind  to  enhancer  
element  and  very  often  more  than  1  factors  to  combined  and  form  co-­‐activators  
 
 

  
Top  sequence  shows  the  promoter  region  and  we  can  see  how  these  proteins  are  binding  different  
parts  of  the  DNA,  meaning  it  will  have  to  fold  according  to  the  binding  sites  of  these  proteins  

 
First  in  search  for  the  TATA  box  within  the  promoter  region,  TBP  recognises  the  TATA  box  and  binds  
to  it,  it  kinks  the  DNA  into  a  right  angle  conformation  that  allows  TFIIB  to  bind
...
 It  then  acts  as  a  recruiter  for  TFIIF  which  associate  
with  the  polymerase  (piggy  bag)
...
 TFIIE  clamps  the  polymerase  into  the  DNA  to  secure  it
...
 dNTPs  and  
ATP  being  converted  to  ADP  are  required  to  start  the  process
...
 When  the  process  has  
kicked  start  and  Pol  starts  travelling  along  DNA,  all  the  transcription  factors  dissociate  apart  from  
TBP  to  keep  DNA  bent
...
 
There  are  however  many  activation  molecules  and  they  all  need  to  bind  at  their  own  regulatory  
sequence  at  the  same  time
...
 Once  all  bound,  it  then  bind  to  the  polymerase  and  start  transcription
...
 They  recognise  the  3’  and  5’  splice  site
...
 A  reaction  takes  place  
and  form  a  lariat  structure
...
   
 
 
 
Every  gene  has  its  own  promoter,  but  the  promoters  may  be  affected  by  the  same  transcription  
factor
...
   

 
By  having  different  promoters  for  separate  genes,  at  different  activator  binding  sites  will  give  
different  levels  of  transcription
...
 All  the  GAL  genes  have  different  set  ups  in  terms  of  where  
the  GAL4  binding  sites  are
...
   
GAL  patheway  –  genes  are  always  switched  off  but  activator  protein  is  always  bound  to  the  cis  
regulatory  element  (UAS)  switching  on  gene  expression
...
 GAL  80  binds  onto  a  region  on  GAL4  protein,  switching  it  off
...
 Gal80  is  a  repressor  
of  GAL4  that  stays  in  the  nucleus  (absent  of  galactose)
...
 Present  of  galactose  cause  a  conformational  change  in  Gal3  and  somehow  it  binds  to  
Gal80  (nucleus/shuttling  back  and  forth?),  but  Gal3  and  Gal80  end  up  in  the  cytoplasm
...
 

 
Gene  Regulation  by  micro  RNA  (miRNA)  Post  transcriptional  regulation  
Regulate  translation,  stop  mRNA  being  read  
to  make  proteins  
Primary  transcripts  of  mRNA  that  fold  into  a  
2nd  structure  (stem-­‐loops)
...
 
Protein  dicer  chop  these  double  strands  into  
separate  strand
...
 Micro  RNA  binds  onto  RISC  
protein  1)  recognise  mRNA  sequence  that  is  not  fully  complementary,  so  close  the  mRNA  with  
loosely  bound  fragments  and  stops  ribosome  binding
...
   

 

  

 



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