Transcript gene regulation

GENE REGULATION
OXYTOCIN
• The following gene is from
mRNA that has been copied
with reverse transcriptase to
produce cDNA
• Sequence provided is from
the coding strand
1 accagtcacg gaccctggac ccagcgcacc cgcaccatgg
ccggccccag cctcgcttgc
61 tgtctgctcg gcctcctggc gctgacctcc gcctgctaca
tccagaactg ccccctggga
121 ggcaagaggg ccgcgccgga cctcgacgtg cgcaagtgcc
tcccctgcgg ccccgggggc
181 aaaggccgct gcttcgggcc caatatctgc tgcgcggaag
agctgggctg cttcgtgggc
241 accgccgaag cgctgcgctg ccaggaggag aactacctgc
cgtcgccctg ccagtccggc
• 301 cagaaggcgt gcgggagcgg gggccgctgc gcggtcttgg
gcctctgctg cagcccggac
• 361 ggctgccacg ccgaccctgc ctgcgacgcg gaagccacct
tctcccagcg ctgaaacttg
• 421 atggctccga acaccctcga agcgcgccac tcgcttcccc
catagccacc ccagaaatgg
• 481 tgaaaataaa ataaagcagg tttttctcct ct
SIGNAL SEQUENCE
• 37-93
• Before signal sequence is the
leader sequence
• Mature Oxytocin 94-120
27 nucleotides = 9 amino acids
• Preproprotein 37- 411
• Signal seq, oxytocin +
neurophysin I Proprotein 94-411
Oxytocin + neurophysin I
• Mature Peptides modified from
Proprotein
• Trailer sequence responsible
for PolyA-polymerase action
1 accagtcacg gaccctggac ccagcgcacc cgcaccatgg
ccggccccag cctcgcttgc
61 tgtctgctcg gcctcctggc gctgacctcc gcctgctaca
tccagaactg ccccctggga
121 ggcaagaggg ccgcgccgga cctcgacgtg cgcaagtgcc
tcccctgcgg ccccgggggc
181 aaaggccgct gcttcgggcc caatatctgc tgcgcggaag
agctgggctg cttcgtgggc
241 accgccgaag cgctgcgctg ccaggaggag aactacctgc
cgtcgccctg ccagtccggc
• 301 cagaaggcgt gcgggagcgg gggccgctgc gcggtcttgg
gcctctgctg cagcccggac
• 361 ggctgccacg ccgaccctgc ctgcgacgcg gaagccacct
tctcccagcg ctgaaacttg
• 421 atggctccga acaccctcga agcgcgccac tcgcttcccc
catagccacc ccagaaatgg
• 481 tgaaaataaa ataaagcagg tttttctcct ct
Notice the 1st 3 nucleotides…
• CODING STRAND
ATG
TEMPLATE STRAND
TAC
mRNA
AUG…start codon
Amino Acid
Methionine (Met)
tgc tac atc cag aac tgc ccc ctg gga
acg atg tag gtc ttg acg ggg gac cct
ugc uac auc cag aac ugc ccc cug gga
Cys-tyr-ile-gln-asn-cys-pro-leu-gly
WOBBLE EFFECT
•
rd
3
base of tRNA may form
H-bonds with more than 1
kind of nucleotide
• Ie AAU and AAC  Asn
TRANSCRIPTION FACTORS
RECOGNIZE SEQUENCES
• Transcription factors
recognize DNA
sequences inorder to
target specific genes
ROLE OF REGULATORY
PROTEINS
• Transcription factors are
genetic switches
• Master Genes ie HOX genes
code for transcription factors
• Related “Regulatory
Proteins” in prokaryotes
PROKARYOTIC REGULATION
• No hormonal regulation
• No introns  no splicing
• No capping/tailing
• Coupled
transcription/translation
• Constitutive – always on;
can be regulated
(enzymes in glycolysis)
• Inducible – off but can be
switched on
• Repressible – on but can
be switched off
OPERONS
• Cluster of genes in which
expression is regulated by operatorrepressor protein interactions,
operator region, and the promoter.
• Promoter
• Repressor
• Operator (controlling site)
• Coding sequences
• Terminator
Lac Operon
• Lactose is an inducer
• “Inducible” operon
• Negative regulation
–-galactosidase (lacZ)
• Breaks lactose into glucose +
galactose.
• Converts lactose to the
allolactose, regulates lac operon.
–Lactose permease (lacY)
• Transports lactose across
cytoplasmic membrane.
–Transacetylase (lacA)
• Positive control when lactose
is E. coli’s sole carbon source
(but not if glucose also is
present).
• Catabolite activator protein
(CAP) binds cAMP, activates,
and binds to a CAP recognition
site upstream of the promoter
(cAMP is greatly reduced in
presence of glucose).
• CAP changes the conformation
of DNA and facilitates binding
of RNA polymerase and
transcription.
• When glucose and lactose are
present, E. coli preferentially
uses glucose due to low levels
of active CAP (low cAMP).
• Regulation of the trp operon:
• “repressible” gene
• 1. Repressor/operator interaction
• When tryptophan is present,
tryptophan binds to trpR gene
product.
• trpR protein binds to the trp
operator and prevents
transcription.
• Is TRP operon neg or pos
regulation?
• What type of regulation
controls
repressors/inducers?
Jacob and Monod
• What is true for E.coli is
also true for the
elephant!
GENETIC SWITCHES
• A switch includes..
a) Binding protein
b) Binding protein recognizes
a strecth of DNA
Significance?
• Mutate the gene encoding
the transcription factor or
the DNA sequence to
which it binds and gene
expression can be
altered!
GENE INACTIVATION
• 1. CHROMATIN structure –
Heterochromatin (tight– gene off)
vs Euchromatin (loose – gene on)
• 2. Methylation – adding methyl
group to inactivate genes on DNA
• 3. small RNA  affects chromatin
structure / interferes with
transcription (RNAi system)
Methylation
• Reinforces inactivation
• Involved in Barr-Body
(inactive X
chromosome)
TRANSCRIPTIONAL CONTROL
• Signals (hormones) in eukaryotes
• Environmental- heat shock proteins
• Regulator proteins (Transcription
factors) – bind to TATA (like
prokaryotes)
• UPEs (Upstream Promoter
Elements) – increase efficiency of
RNA pol.
Example…
• Glucocorticoids released
by stress bind to
steroid receptor (in liver)
 forms complex 
binds to DNA  activates
genes involved in
gluconeogenesis
Weakly transcribed
Strongly transcribed
• TRANSLATIONAL CONTROL
• CAPPING – 7’ methyl guanosine
added to 5’  role in initiation/
protection
• Poly A tail  protection,
promotes transport out of
Nucleus
• Splicing – Introns removed 
Exons for code
• Differential mRNA
processing
–Cells in each tissue
produce own version of
mRNA
–For example, different
forms of troponin, a protein
that regulates muscle
contraction, produced in
different muscle tissue
• POST TRANSLATIONAL
• modification of polypeptide
(ie reduction in size of
proinsulin to insulin)
• chemical modification –
addition of phosphate
(Kinases) removal of
phosphate (Phosphatases)
• Examine the insulin
pathway as a form of
gene regulation