Regulation of Eukaryotic Gene Expression

9/18/2012

Lecture Notes: Dr.Santosh Kumar

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Gene Expression

Constitutive – Genes expressed in all cells (Housekeeping genes) Induced – Genes whose expression is regulated by environmental, developmental, or metabolic signals. signals.

9/18/2012

Lecture Notes: Dr.Santosh Kumar

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Gene Expression
• Gene Expression is a process by which a gene’s DNA sequence is turned into functional protein • Marked by the production of mRNA in a cell • All cells contain the same genome, but not all genes are transcribed & translated • Specific cues to express genes include: temperature changes, nutrients in the external environment, hormones, or other complex signals
9/18/2012 Lecture Notes: Dr.Santosh Kumar
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9/18/2012

Lecture Notes: Dr.Santosh Kumar

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Gene Control
• Gene regulation is a process by which genes are controlled (turned on and off) in response to signals • Cells regulate the genes that are expressed • Prokaryotic & eukaryotic cells regulate a bit differently
• Transcriptional
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regulation (common to both cells) refers to controlling the amount of mRNA transcribed.
Lecture Notes: Dr.Santosh Kumar
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Multilevel Eukaryotic Gene Regulation 1. The types of regulation in eukaryotes 2. Gene structure of eukaryotes 3. Features of Eukaryotic Genomes 4. Multi-level of gene expression and regulation

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Opportunities For The Control Of Gene Expression In The Eukaryotic Cell

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Gene Regulation at DNA Level Chromatin Remodeling 1. Changes of DNA Topo structure Formation of ssDNA DNase I hypersensitive site

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Lecture Notes: Dr.Santosh Kumar

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2. DNA Methylation

DNA Methylation
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 CpG islands ----- are genomic regions that contain a high frequency of CG dinucleotides. ----- CpG islands particularly occur at or near the transcription start site of housekeeping genes.

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Lecture Notes: Dr.Santosh Kumar

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TF

RNA pol Active transcription

Unmethylated CpG island

TF

RNA pol CH3

CH3

CH3

Repressed transcription

Methylated CpG island
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 Housekeeping gene -A gene involved in basic functions is required for the sustenance of the cell. Housekeeping genes are constitutively expressed  Luxury gene - are those coding for specialized functions synthesized (usually) in large amounts in particular cell types.

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3. Histone modification  methylation  acetylation

TF

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4. Changes of nucleosome High Mobility Group (HMG) Protein
    

non-histone chromatin proteins high mobility in PAGE soluble in 2-5% TCA small < 30 kDa high content of charged amino acids: Asp, Glu

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Lecture Notes: Dr.Santosh Kumar

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5. Other gene regulation at DNA Level (1) Gene Deletion (2) Gene Duplication (3) DNA Rearrangement (4) Gene Amplification (5) Chemical Activator (6) Environmental Activator

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Lecture Notes: Dr.Santosh Kumar

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Transcriptional Regulation
• Once the amount of mRNA is controlled, the amount of protein is indirectly controlled • RNA polymerase binds at the promoter (found “upstream” of gene sequences), which indicates the beginning site for transcription • Eukaryotic promoters commonly have a CAAT box (GGCCAATCT) about 80 nucleotides upstream of the start site of gene and a TATA box (TATAAAA) about 30 nucleotides upstream of start site of gene
9/18/2012 Lecture Notes: Dr.Santosh Kumar

• These bind transcription factors

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Molecular Control - Transcription Factors
Transcription factors are DNA binding proteins that regulate the binding of RNA polymerase to stimulate transcription of a gene.

•Accessory proteins for eukaryotic gene expression
•Bind to the TATA & CAAT boxes to stimulate transcription • Bind upstream to either enhance or repress transcription of a gene by assisting or blocking RNA polymerase Lecture Notes:binding Dr.Santosh Kumar

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Basal/General transcription factors
   

Required for transcription initiation Required for proper binding of RNA polymerase to the DNA Each binds to a sequence near promotor Facilitates alignment of RNA polymerase

Special Transcription factors

 
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Increase transcription in certain cells or in response to signals Bind to enhancers Promotor specific (HRE’s for e.g.)
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Transcriptional Regulation

Regulation occurring at the initiation of transcription. transcription. Involves regulatory sequences present within the promoter region of a gene (cis-elements) (cisInvolves soluble protein factors (trans-acting (transfactors) that promote (activators) or inhibit (repressors) binding of the RNA polymerase to the promoter

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Lecture Notes: Dr.Santosh Kumar

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Transcriptional Regulation 1. Cis-acting element (1) What is cis-acting element?  Concept Cis-acting elements - DNA sequences close to a gene that are required for gene expression

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CisCis-elements
  

Typically found in 5’ untranscribed region of the gene (promoter region). region). Can be specific sites for binding of activators or repressors. repressors. Position and orientation of cis element relative to transcriptional start site is usually fixed. fixed.

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Lecture Notes: Dr.Santosh Kumar

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 Features of cis-acting elements

(1) Promoter  Core promoter  in eukaryote: TATA-box, Initiator (Inr)  in prokaryote: -10 region, Inr

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 Proximal elements of promoter  in prokaryote: -35 region  in eukaryote: CAAT-box, GC-box UPE: upstream promoter element UAS: upstream activating sequence (2) Terminator A DNA sequence just downstream of the coding segment of a gene, which is recognized by RNA polymerase as a signal to stop transcription.
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(3) Enhancer A regulatory DNA sequence that greatly enhances the transcription of a gene.

(4) Silencer A DNA sequence that helps to reduce or shut off the expression of a nearby gene.

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(5) Insulators

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No transcription

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The genome is divided into domains by boundary elements (insulators). Insulators can block the spreading of chromatin modifications from one domain to another.
Lecture Notes: Dr.Santosh Kumar

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Enhancers
Enhancers are a class of cis-elements that can be located either cisupstream or downstream of the promoter region (often a long distance away). away).  Enhancers can also be present within the transcribed region of the gene. gene.  Can act over several thousand bp  Function independent of orientation  Function independent of position – upstream, downstream, etc. etc. (different than promotors- close to gene and only one promotorsorientation)  Enhancers can be inverted and still function 5’-ATGCATGC-3’ = 5’-CGTACGTA-3’ ATGCATGCCGTACGTA
9/18/2012 Lecture Notes: Dr.Santosh Kumar
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Enhancers
• Many regulated contain enhancers genes

• They are regulatory DNA sequences, about 50 or more base pairs They are found upstream (occasionally downstream), and can be bound by regulatory proteins

• Can be located far, but a loop in DNA causes contact • The regulatory proteins are called activators
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 

Yellow gene in Drosophila Tissue specific enhancers for pigmentation for each body part Mosaic patterns- alterations in yellow gene patternstranscription in some body parts but not others Also SV40 enhancer (simian virus 40) SV40 40)

9/18/2012

Lecture Notes: Dr.Santosh Kumar

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How do enhancers work?
  

Influence activity of proteins that bind promoters RNA pol and basal transcription factors Physical contact with other proteins?

Enhancer and promotor regions brought together by DNA folding

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Lecture Notes: Dr.Santosh Kumar

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Activators
• Activators are DNAbinding proteins that regulate one or more genes by increasing the rate of transcription •They bind to specific cis-elements particular enhancers • They interact with transcription factors and RNA polymerase to activate or enhance transcription of a gene by promoting assembly of transcription initiation complex • Examples include certain hormones
Lecture Notes: Dr.Santosh Kumar 9/18/2012 •Co-activators bind to proteins associated with cis-elements
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Repressors
• A repressor is a DNA-binding protein that regulates the expression of one or more genes by decreasing the rate of transcription
• Repressors

bind within the promoter to specific cic-elements, preventing RNA polymerase from binding thereby inhibiting assembly of transcription initiation complex. •Co-repressors bind to proteins associated with cis-elements • Identifying promoters, enhancer sequences, and transcription factors is important for making biotech products!
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9/18/2012

Lecture Notes: Dr.Santosh Kumar

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2. What is trans-acting factor?  Concept trans-acting factors - usually they are proteins, that bind to the cis-acting elements to control gene expression.

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These trans-acting factors can control gene expression in several ways:  may be expressed in a specific tissue  may be expressed at specific time in development  may be required for protein modification  may be activated by ligand binding

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(1) RNA polymerase  prokaryotic RNA Pol  eukaryotic RNA Pol

(2) Transcription factors  Basal/general TFs  Specific TFs

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(3) Domains of trans-acting factors  DNA binding domain DBD  Transcription activating domain

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DNADNA-binding and transcription-activation activities are transcriptioncarried by independent domains of an activator. activator. The role of the DNA-binding domain is to bring the DNAtranscriptiontranscription-activation domain into the vicinity of the promoter. promoter.

An activator has independent domains
9/18/2012 Lecture Notes: Dr.Santosh Kumar

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Structural Motifs in DNA-Binding Regulatory Proteins
• Crucial feature must be atomic contacts between protein residues and bases and sugar-phosphate backbone of DNA • Most contacts are in the major groove of DNA • Activators are classified according to the type of DNA-binding domain. • Members of the same group have sequence variations of a specific motif that confer specificity for individual DNA target sites. • 80% of regulatory proteins can be assigned to one of three classes: helix-turn-helix (HTH), zinc finger (Zn-finger) and leucine zipper (bZIP) • In addition to DNA-binding domains, these proteins usually possess other domains that interact with other proteins Lecture Notes: Dr.Santosh Kumar 9/18/2012
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a. HTH (helix-turn-helix)
First identified in 3 prokaryotic proteins two repressor proteins (Cro and cI) and the E. coli catabolite activator protein (CAP) • Arrangement of two α-helices that form a site that binds to DNA, one fitting into the major groove of DNA and the other lying across it. • Two alpha helices separated by a loop with a beta turn • The C-terminal helix fits in major groove of DNA; N-terminal helix stabilizes by hydrophobic interactions with C-terminal helix
9/18/2012 Lecture Notes: Dr.Santosh Kumar
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Helix-turnHelix-turn-helix motifs
Structure: • about 20 amino acids long • 2 short alpha helicies ( 7 – 9 amino acids long) • DNA recognition helix (binds specific DNA sequence) • Recognition helix and 2nd helix form ~ 90° angle • very short turn ( NOT a beta-turn) • Often glycine at start of the turn (helix breaker) • Residues 1-7 of the motif are the first helix (but called "helix 2") • Residue 9 is the turn maker - a Gly, of course • Residues 12-20 are the second helix (called "helix 3") • Recognition of DNA sequence involves the sides of base pairs that face the major groove

9/18/2012

Lecture Notes: Dr.Santosh Kumar

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α-helix (N-terminus)----specific α-helix (C-terminus)----non-specific

GENE
GalR -cro P22-cro Position
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alpha - helix

turn

alpha - helix

A T I K D V A R L A G V S V A T V S R V I N F G Q T K T A K D L G V Y Q S A I N K A I H G T Q R A V A K A L G I S D A A V S Q W K E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
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b. homeodomain --60 aa long DNA binding protein motif α-helix 1 α-helix 2 α-helix 3 ---- DNA major groove

The homeodomain has three helices

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helix turn helix motif: binds to DNA

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c. Leu zipper

 First found in C/EBP, a DNA-binding protein in rat liver nuclei  Now found in nearly all organisms  Characteristic features: a 28-residue sequence with Leu every 7th position and a "basic region"  This suggests amphipathic alpha helix and a coiled-coil dimer  Hydrophobic residues (Leu) align on one side of the helix

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• Leucine zipper proteins dimerize, either as homo- or hetero-dimers • The basic region is the DNArecognition site and binds to DNA. • Basic region is often modeled as a pair of helices that can wrap around the major groove • Homodimers recognize dyadsymmetric DNA • Heterodimers recognize nonsymmetric DNA • Fos and Jun are classic bZIPs
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bZIP – A bZIP (basic zipper) protein has a basic DNA-binding region adjacent to a leucine zipper dimerization motif.
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d. Zinc finger
 First discovered in TFIIIA from Xenopus laevis, the African clawed toad  Now known to exist in nearly all organisms  Two main classes: C2H2 and Cx  C2H2 domains consist of Cys-x2-Cys and His-x3-His domains separated by at least 7-8 aas  Cx domains consist of 4, 5 or 6 Cys residues separated by various numbers of other residues  C2H2 -type Zn fingers form a folded beta strand and an alpha helix that fits into the DNA major groove  Cx-type Zn fingers consist of two mini-domains of four Cys ligands to Zn followed by an alpha helix: the first helix is DNA recognition helix, second helix packs against the first
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Zn fingers form a folded beta strand and an alpha helix that fits into the DNA major groove.

9/18/2012

Lecture Notes: Dr.Santosh Kumar

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ZincZinc-Finger Motifs
Several subtypes (Cys4, Cys2-His2 …) • Example: Cys2 His2 type • Zinc does not interact with DNA • Usually multiple zinc-fingers in a row • At least some also bind RNA • Consensus sequence: [Y,F]-X-C-X2-4-C-XXX-F-XXXXX-L-XX-H-X3-5-H

C C

Zn

H H

C C

Zn

H H

C C

Zn

H H

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e. Helix-loop-helix

helix-loophelix-loop-helix – The motif that is responsible for dimerization of a class of transcription factors called HLH proteins. proteins.

A bHLH protein has a basic DNADNA-binding sequence close to the dimerization motif. motif.

HLH proteins form two sorts of dimers
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f. Bromodomain g. Chromodomain h. Steroid Receptor – Transcription factors that are activated by binding of a steroid ligand.

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Transcription Activation Domains  Acidic domains  Glutamine-rich domains  Proline-rich domains

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Post-Transcriptional Regulation  Control of gene expression usually involves the control of transcription initiation.  But gene expression can be controlled after transcription, with mechanisms such as: 1. Gene Regulation of mRNA Processing exon shuffling alternative gene splicing - recognizes different splice sites in different tissue types. The mature mRNAs in each tissue possess different exons, resulting in different polypeptide products from the same gene.
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9/18/2012

Lecture Notes: Dr.Santosh Kumar

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2. Gene Regulation of mRNA Editing - creates mature
mRNA that are not truly encoded by the genome. For example –  apolipoprotein B exists in 2 isoforms  one isoform is produced by editing the mRNA to create a stop codon  this RNA editing is tissue-specific

3. mRNA Longevity 4. mRNA Transport Control 5. RNA Interference (RNAi)  miRNA  siRNA
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RNA interference involves the use of small RNA molecules The enzyme Dicer chops double stranded RNA into small pieces of RNA  micro-RNAs bind to complementary RNA to prevent translation  small interfering RNAs degrade particular mRNAs before translation

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The left petunia is wild-type; the right petunias contain transgenes that induce suppression of both transgene and endogenous gene expression, giving rise to the unpigmented white areas of the flower.

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What is a microRNA (miRNA)?
Controlling gene expression post-transcriptionally. postmicroRNA is an abundant class of newly identified small nonnoncoding regulatory RNAs.
Major characteristics of miRNAs: • 18-26 nt in length with a majority of 21-23 nt • non-coding RNA • derived from a precursor with a long nt sequence • this precursor can form a stem-loop 2nd hairpin structure • the hairpin structure has low minimal free folding energy (MFE) and high MFE index

Slide courtesy of Baohong Zhang, LectureCarolina Univ Kumar East Notes: Dr.Santosh 9/18/2012
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miRNA regulates plant development
miRNA 156 increasing leaf initation, decreasing apical dominance, and forming bushier plant. miRNA 164 stamens are fused together.

miRNA 172 sepal and petal disappeared.

miRNA 319 Leaf morphology
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WT

Lecture Notes: Dr.Santosh miRNA Slide courtesy ofKumar Baohong Zhang, East Carolina Univ

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Cytoplasmic Control

mRNA stability: stability:
Long vs. short lived mRNAs vs.  Long- many rounds protein synthesis from one LongmRNA  Short – rapidly degraded, needs more transcription to replenish (half-life) (half

 

Rapid mRNA degradation may be desirable HalfHalf-life problem with making a drug, too

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mRNA Stability
  

Poly A tails – can add stability Longer tails stabilize message more E.g., histone mRNAs no poly A tails; message tails; very short lived

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Induction of Transcription

Not found as often in eukaryotes as in prokaryotes Induction can work by: by:
Temperature  Light  hormones

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Induction of Transcription

Temperature
Synthesize heat shock proteins (HSPs)  Transcriptional regulation – stress of high heat signals HSPs to be transcribed  Studied in Drosophila- but occurs in humans also Drosophila

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Induction of Transcription

Light
RBC (ribulose 1,5 bisphosphate carboxylase) carboxylase)  Plants must absorb light energy  RBC produced when plants are exposed to light

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Induction of Transcription

Hormones
Secreted, circulate through body, make contact with target cell and regulate transcription  Called signal molecules  2 classes of hormones that activate transcription

Steroid hormones  Peptide hormones

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Steroid Hormones
  

Small, lipid molecules derived from cholesterol Easily pass through cell membranes Examples
Estrogen  Progesterone  Testosterone  Glucocorticoids

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Steroid Hormones

HRE’sHRE’s- hormone response elements – mediate hormone induced gene expression Number of HRE’s dictate strength of response (work cooperatively)

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Peptide Hormones
 

Linear chain of amino acids Examples  Insulin  Growth hormone  prolactin

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Peptide Hormones

Cannot pass through cell membrane easily, so convey signals through membrane bound receptors Signal transduction – hormone binds receptor on cell surface, signal gets internalized, then cascade of events begin

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Translational and Post-translational Regulation 1. Translation Control Blocking mRNA Attachment to Ribosomes 2. Regulation of Protein Processing Protein Modification

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 Amino acid residues phosphorylated
 Ser/Thr type  Tyr type

 Catalysis Features of Actions
 Reversible  Integrated signals from different pathways effectively  The same kind kinase or phosphatase is multible-substrates.  modified different amino acids, different influences

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3. Regulation of Protein Stability

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Summary
1. Genomic structure of eukaryotes 2. Regulation levels of eukaryotic gene expression. 3. Regulation at DNA level 4. Regulation at transcritional level 5. Transcriptional regulation 6. The mechanism of RNAi 7. Translational and post-translational regulation: 8. Protein phosphorylation and its mechanism

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9/18/2012

Lecture Notes: Dr.Santosh Kumar

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