Regulation of Cellular Functions (a.k.a. Gene Expression)
Transcription
start
5’ Regulatory Region
Enhancer
variable
TF
Promoter
Element
-1kb to -40
TATA
Box
Exon 1
-30
3’ Region
Transcribed Region
0
Exon 2
intron 1
Exon 3
intron 2
TRANSCRIPTION
TF
Transcription factors
Primary Transcript
Exon 1
5’
Exon 2
Exon 3
3’
intron 2
intron 1
RNA
PROCESSING
Cap
mRNA
poly A tail
5’ mG
5’ Untranslated
Region (UTR)
Exon 1
Exon 2
AAAAA 3’
Exon 3
3’ UTR
coding sequence
ATG
(start codon)
stop
codon
TRANSLATION
Degradation
NH3
Protein
Degradation
COOH
POSTTRANSLATIONAL
MODIFICATION
Degradation
FUNCTION
Genomic equivalence
Different cells perform different functions.
The function of a cell depends on the sum total of the proteins that are present in that
cell.
How can 2 cells in an organism express 2 different sets of proteins.
1. differential loss of genetic material during cell differentiation
2. differential expression of genes in a complete genome.
Evidence for genomic equivalence: cloning
Take a differentiated cell and regenerate a complete organism—shows the cell was
totipotent and contained the full set of genetic information to make the entire
organism. Therefore, only a subset of the genetic information is expressed in the
differentiated cell.
So cell differentiation depends on differential gene regulation and to understand how a
cell becomes differentiated, we have to understand how gene expression is regulated.
Regulation of Cellular Functions (a.k.a. Gene Expression)
RNA Regulation
Transcription – DNA/protein interactions
Cis elements—DNA sequences that transcription factors bind to regulate gene
transcription
-most commonly found in the 5’ region of a gene but can be in 3’ or even
transcribed regions.
Trans factors (transcription factors)—proteins that bind cis elements and regulate
gene transcription
Activators or Repressors
Processing
Capping & polyadelylation
Intron splicing
Degradation
UTR
Micro RNAs
Localization
UTR
Translation
UTR
Micro RNAs
Key RNA techniques
1. Northern (or RNA gel) blotting
2. RT-PCR (reverse transcription-polymerase chain reaction)
3. Microarray
4. In situ hybridization
5. Reporter genes (promoter fusions)
1-4 show steady-state RNA levels (transcription + turnover)
1-3 show average levels among cells included in the RNA extraction process
4,5 show cellular distribution of transcript
Protein Regulation
Translation
Ribosomes / Initiation and elongation factors
Post-translational modifications
Subcellular targeting & localization
Interactions with other proteins
Interactions with cofactors (GTP, cAMP, ions, etc.)
Phosphorylation / dephosphorylation
Glycosylation
Lipidation
Oxidation/reduction
Cleavage
Degradation
Ubiquitination / Proteosome
Key protein techniques
1. Western (immunoblot)
2. Immunolocalization (immunohistochemistry or immunocytochemistry)
3. Reporters / translational fusions
Examples
Sxl and sex determination (splicing)
Bicoid (mRNA transport, localization, translational control)
Easter-Toll-Dorsal (proteolytic activation, protein interaction-receptor conformational
change, protein phosphorylation, ubiquitination, nuclear translocation)