Transcript Regulation of gene expression

Regulation
of gene expression
Kamila Balušíková
• Gene expression vs. Regulation of gene expression
• Unicellular organisms: requirements for adaptation to
changed environmental conditions.
• Multicellular organisms: requirements for the selective
expression of genes → relevant differentiation status of
various cell types
► cell differentiation arises because cells make and
accumulate different sets of RNA and protein
molecules; that is, they express different genes
• Whether a gene is expressed or not
depends on a variety of factors:
 the type of cell
 its surroundings
 its age
 extracellular signals
 each cell contains complete genome
but express only part of the genes
 THE NEED OF REGULATION
 cells have to be able to respond to changes in
environment
 THE NEED OF REGULATION
(B)
Levels of the regulation of GE
• Genome (DNA)
• Transcription (DNA → RNA/primary transcript) –
transcriptional control
• Posttranscriptional modifications (RNA/primary
transcript → mRNA) – processing control, RNA transport
and localization control
• Translation (mRNA → polypeptide chain) – translational
control, mRNA degradation control
• Posttranslational modifications (polypeptide chain →
functional protein)
• Protein degradation (functional protein → decomposed
protein) – protein activity control
Regulation of GE
Regulation of transcription – most economic
X
Regulation of protein activity – fastest
Genome (DNA)
Regulation of the access to genomic
DNA
– Condensation/decondensation of
the chromosome
the higher level of DNA condensation
the less DNA accessibility for
transcription factors and RNA
polymerases
– Structure of chromatin (heterochromatin x
euchromatin
X inactivation
 X inactivation (always only 1 X
chromosome is active - Xa, Xi)
 Non-translated (non-coding)
RNA – Xist (X-inactivation
specific transcript), Xi is coated
by Xist RNA which is
transcribed by this Xi → if the
XIC is spread over the entire
chr. – inactivation of this chr.)
 Limiting blocking factor – binds
to X inactivation center (XIC)
and it blocks inactivation (only
in 1 Xa)
 Xa – no coating by Xist, no
inactivation
Position effect
(PEV – position effect variegation)
• Each chromosome (and gene) has its own place in
nucleus (sufficient amount of RNA polymerase,
regulation factors, euchromatin)
• Difference in the gene expression which depends on
the position of his gene in genome = position effect
• Experiment with drosofila:
white gene (color of the eye)
if moved near the
heterochromatin
– inactivation of the gene)
Acetylation of histones
• Histone acetylation –
histon acetylase
→ activation
• Histone deacetylase
removes the acetyl
group from histone
→ DNA less accessible
Methylation
• Genes which are not expressed can be methylated (→ their
expression is blocked)
• Enzyme methylase - catalyzes
the methylation of cytosine in
DNA (5-methylcytosine)
• Methylation of CG sites
• (sequence: -C*G-p-C*G-)
• The degree of DNA methylation corresponds to the degree of
gene expression (methzlated gene = non-expressed gene)
• Genomic imprinting – only one copy of gene (maternal x
paternal) is active
Epigenetics
• changes in phenotype or gene expression
caused by mechanisms other than changes in
the underlying DNA sequence
• Epigenetics processes are usually caused by
transcription repression which is controlled by
chromatin modulation
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–
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–
Genomic imprinting
Position effect
Gene dose compensation (X inactivation)
Chemical modification of DNA, histones
Chromatin remodeling
Transcription
• Controlled by the binding of specific regulatory proteins to
specific regulatory DNA sequences - Based on protein-DNA
interaction (DNA binding proteins)
• Most economic way of regulation
• Eucaryotic cells: each gene under individual control,
complex regulation
• Procaryotic cells: regulation of transcription of whole operon,
simple regulation
• Repressors/activators bind to specific promoter
sequences
DNA binding proteins recognize sequences, usually bind to
the major groove
• Regulatory DNA sequences
are needed in order to switch the gene on or off
procaryotes – 10 nucleotide pairs short (simple gene
switches)
eucaryotes – 10 000 nucleotide pairs away (microprocessors)
–
• Gene regulatory proteins
– bind to regulatory DNA sequences
 The combination of a DNA sequence and its associated
protein molecules acts as the switch to control transcription
 single contact = a non-covalent contact between one base
pair and amino acid
 Regulatory protein contains 10-20 such specific contacts
 DNA - protein motifs = general folding patterns of regulatory
proteins
DNA - protein motifs = general folding
patterns of regulatory proteins
Types of DNA
binding proteins:
1.
2.
3.
4.
Leucine zipper
Helix-loop-helix
Helix-turn-helix
Zinc finger
Zinc finger
Zn atom - stabilization
type helix + β sheet
helix + helix (dimerization)
Helix-loop-helix
• Longer loop – position of helix is
not fixed
• Dimerization (homo and
heterodimer)
• Regulation – dimerization with
shorter protein
Leucine zipper
Dimerization (hydrofobic interaction of leucins)
of α helixes
Helix-turn-helix
Short loop, fixed angle
C terminal helix: binding to
major groove
Procaryotes
• effective response to quickly changing physical /chemical
conditions of environment
• main purpose: survival of the cell
• regulation especially on the transcription level
• very short lifetime of mRNA (cca 3 min.)
• polygenic mRNA
• Regulation of gene expression best studied in proteins
with enzymatic function
Regulation of transcription in procaryotes
• OPERON – transcription unit , a cluster of genes on the
chromosome , which are regulated by a single promoter
and operator, they are transcribed as one long mRNA
molecule
– 1 mRNA (with several genes) = 1 transcription unit
– polycistronic transcript
• PROMOTER – initiation site, where transcription actually
begins, upstream region, a sequence which contains
sites that are required for the RNA polymerase to bind to
the promoter
• OPERATOR – a short DNA sequence (cca 15
nucleotides in length) within the promoter which is
recognized by a gene regulatory proteins
• regulatory gene – is localized outside the operon,
codes for regulatory protein, its expression is usually
constitutive and controlled by its own promoter
• regulatory proteins – bind to the promoter/operator,
(encoded by regulatory gene)
– Repressor protein - switches genes off, represses
them (synthesis of tryptophan)
– Activator protein - switches genes on, activates
them (degradation of sugars, CAP)
Regulation of transcription in procaryotes
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•
•
Simple regulation
Specific sigma factors (interaction with RNA polymerase)
Regulatory proteins: activators, repressors
Trp operon
Tryptophan repressor
Regulation of transcription in eucaryotes
Eucaryotes x procaryotes
• RNA-polymerase → procaryotes 1 x eucaryotes 3
RNA pol I (rRNA), II (mRNA), III (tRNA, small RNA)
• Eucaryotic RNA-polymerase needs a large set of proteins general transcription factors - for initiation of
transcription
(they assemble at the promoter (TATA box), they are
highly universal, evolutionary conserved)
• Eucaryotes can influence the initiation of transcription by
specific transcription factors (repressors and activators)
even when they are bound to DNA thousands of nucleotide
pairs away from the promoter, this feature allows a single
promoter to be controlled by an almost unlimited number of
regulatory sequences scattered along the DNA
• the DNA, at this time, loops out to allow all proteins to
come into contact, regulation is complex - combinatorial
control
• Eucaryotic transcription initiation must take account of the
packing of DNA into nucleosomes and more compact forms
of chromatin structure
TATA box is highly conserved
promoter in eucaryotes
Preinitiation complex
and initiation of transcription
RNA polymerase II preinitiation complex
• presence of general transcription factors (TFII) are needed
for assembly of Pol II at the DNA
• TFII create multimers and highly conserved
• Proteins (TFII) are bound in specific order and create RNA
pol II preinitiation complex
TBP binds to TATA-box
and folds DNA double helix
TBP is subunit of TFIID
Initiation of transcription
General transcription factors are usually not sufficient, the
presence of activator proteins is needed (they help to assemble
general transcription factors and RNA pol. into initiation complex) x repressors
slow down, block this process
• enhancer – binding site for activator protein
•
Complex of regulatory proteins
– combination of the effect of several regulatory proteins
Combinatorial control
• groups of proteins work together to determine the
expression of a single gene
• The effects of multiple gene regulatory proteins combine
to determine the rate of transcription initiation
• The effect of a single gene regulatory protein can be
decisive in switching any particular gene on or off
• This do so by completing the combination
Activators
Synergic effect
Often in distant regions
Activator can be also a repressor
Acetylation of histones
Repressors
Repressors can operate in
different ways
Repressors and activators attract histone acetylases and
deacetylases
Control of regulatory proteins
Some ways in which the activity of gene regulatory proteins is regulated
in eucaryotic cells
Posttranscriptional modifications
• RNA capping and RNA
polyadenylation increase the stability
of mRNA
• Alternative splicing
• RNA editing
Alternative splicing
• the primary transcript can be spliced in various ways,
removing not only all introns but also certain exons,
to produce different mRNAs, depending on the cell type
in which the gene is being expressed, or the stage of
development of the organism
– enables eucaryotes to increase the coding potential of
their genomes
– creates plasticity of eucaryotic genetic information
– influence of environmental protein factors on RNA
splicing
– allows different proteins to be produced from the
same gene (1 gene – 5 proteins)
– alternative splicing is tissue specific
RNA editing
• RNA editing - insertion or deletion of nucleotides
or substitution of nucleotides in transcribed RNA
– certain change of transcribed genetic information
– it can result in the appearance of new initiation
and stop codons
– gRNA (guide RNA)
RNA editing
Translation
• Each mRNA molecule is eventually degraded (RNA
degradation) within the cell
• The lifetime of corresponding mRNA affects the
expression of particular gene (longer lifetime of the mRNA
means higher level of translated protein and vice versa)
• The lifetime of mRNA is regulated by nucleotide
sequences in the 3' untranslated region of mRNA
• Translation can be also regulated by specific protein
binding to mRNA
– eg. IRP (iron regulatory protein)/IRE (iron responsive element)
system
Regulation on the principle of stabilization
of mRNA
Iron-dependent regulation of the stability of transferrin-receptor mRNA
system IRP/IRE
Regulation on the principle of inhibition
of translation
Iron-dependent regulation of translation of ferritin mRNA
system IRP/IRE
Posttranslational modifications
Newly synthesized polypeptide chain can be modified in
several ways including both the cleavage of polypeptide
chain and the binding of molecules
– Removal of methionine from the N end: every newly
synthesized polypeptide chain starts with methionine
– Removal of single sequence: the signal sequence is a
sequence of aminoacids serving as a signal for transfer to
required location
– Proteolytic cleavage: the formation of functional protein
by cleavage of a precursor polypeptide chain (proinsulin
→ insulin)
– Formation of disulfide bonds: they are formed
between adjacent cysteines. They help to stabilize
protein structure
– Chemical modification of amino acids:
• phosphorylation (binding of phosphate)
• hydroxylation (binding of –OH group)
– Glycosylation: the binding of oligosacharide chains
(glycoproteins)
– Binding of prosthetic groups: the binding of
prosthetic group (nonamino acid/nonprotein molecule)
can be required for the functioning of protein (heme in
hemoglobin)
Insulin
Protein degradation - proteasome
• Protein degradation – regulation of the amount of particular protein
within the cell
• Individual proteins vary in their life span
• Most proteins in cells are degraded by proteasomes
- Proteasome is a large complex of proteolytic enzymes forming
kind of cylinder
- Proteins are marked for degradation by the covalent binding of a
small protein ubiquitin