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REGULATION OF GENE EXPRESSION IN
PROKARYOTES & EUKARYOTES
Lecture By
RAVI DHANDE
Lecturer,Department of BotanyShri Shivaji College, [email protected]
REGULATION OF GENE EXPRESSION IN
PROKARYOTES & EUKARYOTES
Lecture By
RAVI DHANDE
Lecturer,Department of BotanyShri Shivaji College, [email protected]
REGULATION OF GENE EXPRESSION IN PROKARYOTESREGULATION OF GENE EXPRESSION IN PROKARYOTES
“NOT ALL THE GENES EXPRESS AT ONE TIME”
Regulated genes
Control cell growth and cell division.Expression is regulated by the needs of the cell and the environment as needed (not continuously).
Constitutive genes
Housekeeping genes Continuously expressed.
Central theme ofCentral theme of protein synthesisprotein synthesis
Operon - what is it?
Cluster of genes in which expression is regulated by operator-repressor protein interactions, operator region, and the promoter.
Contents of an operon:
PromoterRegulatorOperator (controlling site)Coding sequences (Adjacent polycystronic coding sequences/ polygenic mRNA)
Terminator
Inducer , Induction? Inducible gene?Repressor, Repression?Repressible gene?
Organization of an inducible gene containing an operon
E. coli lac operonFrancois Jacob and Jacques Monod (Pasteur Institute, Paris, France)
E. coli expresses genes for glucose metabolism continuously.
Metabolism of other alternative types of sugars (e.g., lactose) are regulated specifically.
Lactose = disaccharide (glucose + galactose), provides energy.
Lactose acts as an inducer (effector molecule) and stimulates expression of three proteins at 1000-fold increase:
-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)•Function is not understood.
General organization of the lac operon of wild-type E. coli.
Order of controlling elements and genes:
lacI: promoter-lacI-terminator
operon: promoter-operator-lacZ-lacY-lacA-terminator
Organization of Organization of laclac operon size operon size
mRNAmRNA Lac iLac i Lac ZLac Z Lac YLac Y Lac ALac A
PolypeptidePolypeptide 38 kd38 kd 116 kd116 kd 30 kd30 kd 30 kd30 kd
Active Active proteinprotein
TetramerTetramer
152 kd152 kd
TetramerTetramer
500 kd500 kd
MonomerMonomer
30 kd30 kd
DimerDimer
60 kd60 kd
FunctionFunction RepressorRepressor B-B-galactosidasegalactosidase PermeasePermease TransacetylaseTransacetylase
Functional state of the E. coli lac operon in the absence of lactose
Functional state of the E. coli lac operon growing on lactose
Mutational study
Different types of mutations occur in lacO, lacI, and promotor:
lacO -change repressor binding site (repressor does not bind)
-continuously expressed
lacI -change repressor conformation (cannot bind operator)
-continuously expressed
promoter -alter affinity for RNA polymerase-increase or decrease transcription rate
Translation of lac operon in wild type and mutant E. coli
Positive control also occurs in the lac operon
Positive control occurs when lactose is sole carbon source for E. coli (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. cAMP level falls down).
• Adding cAMP to cells restore transcription of the lac operon even when glucose is still present.
Lac operon, +ve gene regulation
• lac promoter begins at -84 bp ends at -8 bp
• CAP-cAMP binding site occurs at -54 to -69.
• RNA polymerase binding site spans -47 to -8.
• Operator is next to the promoter at -3 to +21.
• mRNA transcript begins at +1 bp within the operator.
• -galactosidase gene has a leader sequence before the start codon.
• -galactosidase start codon (AUG) is at position +38 to +40
Base pair sequence of controlling sites, promoter, and operator for lac operon of E. coli.
Positive control of the lac operon
glucose
glucose cAMP
cAMP-CAP
cAMP –CAP-RNA POL COMPEX
Gene turn on
cAMP
NO cAMP –CAP
NO cAMP –CAP-RNA POL COMPEX
Gene turn off
REGULATION OF GENE EXPRESSION IN EUKARYOTESREGULATION OF GENE EXPRESSION IN EUKARYOTES
Central theme ofCentral theme of protein synthesisprotein synthesis
Levels of Eukaryotic Gene Levels of Eukaryotic Gene RegulationRegulation
Transcription
RNA processing
mRNA transport
mRNA translation
mRNA degradation
Protein degradation
Acetylation / deacetylation of histoneAcetylation / deacetylation of histone
Acetylation of N-terminal end of H3 protein specificallyAcetylation of N-terminal end of H3 protein specifically
Lysine residue.Lysine residue.
Acetylation leads to gene expression by Acetylation leads to gene expression by Acetyl trasferase Acetyl trasferase
Deacetylation leads to gene repression byDeacetylation leads to gene repression by deacetylase deacetylase
AcetylationAcetylation
DeacetylationDeacetylation
DNA methylation and transcription control
Methylation occurs most often in symmetrical CG sequences.
Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes.
Methylation leads to suppression of genes.
Demethylation leads to expression of genes.
Hormone for regulation of transcription
Cells of higher eukaryotes are specialized and generally shielded from rapid changes in the external environment.
Hormone signals are one mechanism for regulating transcription in response to demands of the environment.
Hormones deliver signals in two different ways:
Steroid hormones pass through the cell membrane and bind cytoplasmic receptors, which together bind directly to DNA and regulate gene expression.
Polypeptide hormones bind at the cell surface and activate transmembrane enzymes to produce second messengers (such as cAMP) that activate gene transcription.
RNA processingRNA processing
Polyadenylation- addition of poly A tail Polyadenylation- addition of poly A tail
Splicing- removal of introns from mRNA.Splicing- removal of introns from mRNA.
I I
E E E
AAAAAAAAA 3’5’
mRNA transport control
Eukaryotic mRNA transport is regulated.
Some experiments show ~1/2 of primary transcripts never leave the nucleus and are degraded.
Mature mRNAs exit through the nuclear pores.
mRNA translation control
Stored mRNAs are protected by proteins that inhibit translation.
Poly(A) tails promote translation.
Stored mRNAs usually have short poly(A) tails(15-90 As vs 100-300 As).
Specific mRNAs are marked for deadenylation (“tail-chopping”)
Activation occurs when create a full length poly(A) tail.
AAAA AAAAAAAAAAA
TranslatedUntranslated
mRNA degradation control
All RNAs in the cytoplasm are subject to degradation.
tRNAs and rRNAs usually are very stable; mRNAs vary considerably (minutes to months).
Stability may change in response to regulatory signals and is thought to be a major regulatory control point.
Various sequences and processes affect mRNA half-life:
• Secondary structure• AU-rich elements• Deadenylation enzymes remove As from poly(A) tail• 5’ de-capping• Internal cleavage of mRNA and fragment degradation
Post-translational control - protein degradation
Proteins can be short-lived or long-lived
Protein degradation in eukaryotes requires a protein co-factor that binds to proteins and identifies them for degradation by proteolytic enzymes.
Amino acid at the N-terminus is correlated with protein stability and determines rate of co-factor binding.
Arg, Lys, Phe, Leu, Trp 1/2 life ≤3 minutes
Cys, Ala, Ser, Thr, Gly, Val, Pro, Met 1/2 life ≥ 20 hours
