eukaryotic gene regulation models (by np mendez)
TRANSCRIPT
Prepared by: Prepared by:
NOE P. MENDEZNOE P. MENDEZMaster of Science in BiologyMaster of Science in Biology
CENTRAL MINDANAO UNIVERSITYCENTRAL MINDANAO UNIVERSITY
EUKARYOTIC GENE REGULATION MODELS
Overview Eukaryotic Gene Regulation Models
A. Gene Expression B. Initiation of Transcription C. Posttranscriptional control D. Posttranslational control
What is a gene? “The entire nucleic acid sequence that is
necessary for the synthesis of a functional polypeptide or RNA molecule.”
Basic structure a gene
Basic structure a gene
Kadonaga (2004)
Eukaryotic Promoter Elements
Kadonaga (2004)
How does an individual cell specify which of its many thousands of
genes to express?
As animal develops, cell types become different from one another, eventually leading to the wide
variety of cell types seen in the adult.
How are genes turned on & off in eukaryotes?How do cells with the same genes differentiate to perform completely different, specialized functions?
– multicellular– evolved to maintain constant internal conditions
while facing changing external conditions– regulate body as a whole
• growth & development– long term processes
• specialization– turn on & off large number of genes
• must coordinate the body as a whole rather than serve the needs of individual cells
Gene expression of eukaryotic cells All organisms must regulate which genes are
expressed at any given time.– They must continually turn genes on and off in response to
external stimuli/signals. In multicellular organisms, regulation of gene
expression is essential for cell specialization.
Gurdon, J.B. 1968. Sci. Am. 219(6): 24–35
In prokaryotic cell – Operon systems,
negative feedback
In eukaryotic cell – regulated at any stage,
noncoding RNAs
Regulation of Gene Expression
Activation of gene structure
Initiation of transcription
Control of gene expression at any stage:
Overview Eukaryotic Gene Regulation Models
A. Gene Expression B. Initiation of Transcription C. Posttranscriptional control D. Posttranslational control
• chromatin changes
• transcription
• processing RNA
• transport to cytoplasm
• degradation of mRNA
• translation
• cleavage, chemical modification
• protein degradation
Complicated regulation system
Binding may form the biochemical basis of translational synergy between cap structure and poly (A) tail
(Preiss & Hentze, 1999).
Stages in gene expression in eukaryotic cell
•Signal
•NUCLEUS•Chromatin
•Chromatin modification:DNA unpacking involvinghistone acetylation and
DNA demethylation•DNA
•Gene
•Gene availablefor transcription
•RNA •Exon•Primary transcript
•Transcription
•Intron•RNA processing
•Cap•Tail
•mRNA in nucleus
•Transport to cytoplasm
•CYTOPLASM•mRNA in cytoplasm
•Translation•Degradationof mRNA
•Polypeptide•Protein processing, such
as cleavage and chemical modification
•Active protein•Degradation
of protein•Transport to cellular
destination
•Cellular function (suchas enzymatic activity,
structural support)
How do you fit all that DNA into
nucleus?
How do you fit all that DNA into nucleus?
– DNA coiling & folding• double helix• nucleosomes• chromatin fiber• looped domains• chromosome
•from DNA double helix to condensed chromosome
DNA of Eukaryotic cells is packaged in chromatin.
Heterochromatin is highly condensed -
transcriptional enzymes can not reach the DNA
Genes within highly packed heterochromatin are
usually not expressed
Acetylation / deacetylation of histones
Methylation [cytosin] - inactive DNA is highly
methylated
1. Chromatin changes
DNA methylation
- Essential for long-term inactivation of genes
during cell differentiation
Gene imprinting in mammals
- Methylation constantly turns off the maternal/
paternal allele of a gene in early development
- certain genes are expressed in a parent-of-
origin-specific manner
Epigenetic inheritance
1. Chromatin changes
• Remember, DNA in eukaryotes packs into
CHROMATIN.• HISTONES form the
NUCLEOSOME, which DNA loops around.
• EUCHROMATIN - less compact; actively
transcribed• HETEROCHROMATIN -
more compact; transcriptionally
inactive.– Heterochromatin can be
either constitutive or facultative.
Chromatin
Histone Modifications In histone acetylation, acetyl groups are attached to
positively charged lysines in histone tail– Acetylation promotes initiation of transcription.
Deacetylation does not This loosens chromatin structure, thereby promoting
the initiation of transcription The addition of methyl groups (methylation) can
condense chromatin The addition of phosphate groups (phosphorylation)
next to a methylated amino acid can loosen chromatin
HISTONES in transcriptionally active genes are often ACETYLATED.
Acetylation is the modification of lysine residues in histones.– Reduces positive charge, weakens the interaction
with DNA.– Makes DNA more accessible to RNA polymerase II
Enzymes that ACETYLATE HISTONES are recruited to actively transcribed genes.
Enzymes that remove acetyl groups from histones are recruited to methylated DNA.
Histone Acetylation
Amino acidsavailable
for chemicalmodification
Histone tails
DNA double
helix
Nucleosome(end view)
(a) Histone tails protrude outward from a nucleosome
Unacetylated histones Acetylated histones(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription
2. Transcription Initiation
Control regions on DNA– promoter
• nearby control sequence on DNA• binding of RNA polymerase & transcription factors• “base” rate of transcription
– enhancer• distant control
sequences on DNA• binding of activator
proteins• “enhanced” rate (high level)
of transcription
Eukaryotic gene and transcript
•Enhancer(distal control
elements)•DNA
•Upstream •Promoter
•Proximalcontrol
elements•Transcription
start site•Exon •Intron •Exon •Exon•Intron
•Poly-Asignal
sequence•Transcription
terminationregion
•Downstream
•Enhancer(distal control
elements)•DNA
•Upstream •Promoter
•Proximalcontrol
elements•Transcription
start site•Exon •Intron •Exon •Exon•Intron
•Poly-Asignal
sequence•Transcription
terminationregion
•Downstream•Poly-Asignal
•Exon •Intron •Exon •Exon•Intron
•Transcription
•Cleaved3 end ofprimary
transcript
•5•Primary RNA
transcript(pre-mRNA)
•Enhancer(distal control
elements)•DNA
•Upstream •Promoter
•Proximalcontrol
elements•Transcription
start site•Exon •Intron •Exon •Exon•Intron
•Poly-Asignal
sequence•Transcription
terminationregion
•Downstream•Poly-Asignal
•Exon •Intron •Exon •Exon•Intron
•Transcription
•Cleaved3 end ofprimary
transcript
•5•Primary RNA
transcript(pre-mRNA)
•Intron RNA
•RNA processing
•mRNA
•Coding segment
•5 Cap •5 UTR•Startcodon
•Stopcodon •3 UTR
•3
•Poly-Atail
•P•P•P•G •AAA AAA
DNA
Activationdomain
DNA-bindingdomain
ActivatorsDNA
Enhancer Distal controlelement
PromoterGene
TATA box
ActivatorsDNA
Enhancer Distal controlelement
PromoterGene
TATA boxGeneral
transcriptionfactors
DNA-bendingprotein
Group of mediator proteins
ActivatorsDNA
Enhancer Distal controlelement
PromoterGene
TATA boxGeneral
transcriptionfactors
DNA-bendingprotein
Group of mediator proteins
RNApolymerase II
RNApolymerase II
RNA synthesisTranscription
initiation complex
Overview Eukaryotic Gene Regulation Models
A. Gene Expression B. Initiation of Transcription C. Posttranscriptional control D. Posttranslational control
3. Processing RNA
•Post-transcriptional modifications
•Alternative RNA splicing •The same primary transcript, but different
the mRNA molecule / exons and introns
4. Regulation of mRNA degradation
•Lifespan of mRNA is important for protein synthesis
•Enzymatic shortening Life span of mRNA determines amount of protein
synthesis– mRNA can last from hours to weeks
Activation or inactivation of protein factors to initiate
translation
4. Regulation of mRNA degradation
RNA interference
Small interfering RNAs (siRNA)– short segments of RNA (21-28 bases)
• bind to mRNA• create sections of double-stranded mRNA• “death” tag for mRNA
– triggers degradation of mRNA
– cause gene “silencing”• post-transcriptional control• turns off gene = no protein produced
•NEW!
•siRNA
Action of siRNA
siRNAdouble-stranded miRNA + siRNA
mRNA degradedfunctionally turns gene off
Hotnew topicin biology
mRNA for translation
breakdownenzyme(RISC)
dicerenzyme
Capping
p-p-p-N-p-N-p-N-p….
p-p-N-p-N-p-N-p…
G-p-p-p-N-p-N-p-N-p…
CH3
G-p-p-p-N-p-N-p-N-p…CH3 CH3
GMP mCE (another subunit)
Capping enzyme (mCE)
methyltransferasesS-adenosyl methionine
The capping enzyme
A bifunctional enzyme with both 5’-triphosphotase and guanyltransferase activities
In yeast, the capping enzyme is a heterodimer In metazoans, the capping enzyme is
monomeric with two catalytic domains.
Capping mechanism in mammals
DNA
Growing RNA
Capping enzyme is allosterically controlled by CTD domains of RNA Pol II and another
stimulatory factor hSpt5
Polyadenylation
Poly(A) signal recognition Cleavage at Poly(A) site Slow polyadenylation Rapid polyadenylation
• G/U: G/U or U rich region
• CPSF: cleavage and
polyadenylation specificity factor
• CStF: cleavage stimulatory factor• CFI: cleavage
factor I• CFII: cleavage
factor II
PAP: Poly(A) polymerase
CPSF
PAP
PABPII- poly(A) binding protein II
PABP II functions:
1.rapid polyadenylation
2.polyadenylation termination
pp
Pol II
ctd
mRNA
PolyA – binding factors
Link between polyadenylation and transcription
Pol II gets recycled
mRNA gets cleaved and polyadenylated
degradation
cap
polyA
cap
splicing,nuclear
transport
pp
aataaa
FCP1 Phosphatase removes phospates
from CTDs
cap
Splicing
The size distribution of exons and introns in human, Drosophila and C. elegans genomes
Consensus sequences around the splice site
YYYY
Molecular mechanism of splicing
Additional factors of exon recognition
•ESE - exon splicing enhancer sequences
•SR – ESE binding proteins
•U2AF65/35 – subunits of U2AF factor, binding to pyrimidine-rich regions and 3’ splice site
Binding of U1 and U2 snRNPs
Binding of U4, U5 and U6
snRNPs
The essential steps in splicing
Rearrangement of base-pair
interactions between snRNAs, release of U1 and
U4 snRNPs
The catalytic core, formed by U2 and
U6 snRNPs catalyzes the first transesterification
reaction
Further rearrangements between U2, U6 and U5 lead to
second transesterification
reaction
The spliced lariat is linearized by debranching enzyme and further degraded in exosomes
Not all intrones are completely degraded. Some end up as functional RNAs, different from mRNA
pp
Pol IIctd
mRNA
SCAFs: SR- like CTD – associated
factorscap
SRssnRNPs
Intron
Co-transciptional splicing
Self-splicing introns
Under certain nonphysiological conditions in vitro, some introns can get spliced without aid of any proteins or other RNAs
Group I self-splicing introns occur in rRNA genes of protozoans
Group II self-splicing introns occur in chloroplasts and mitochondria of plants and fungi
Group I introns utilize guanosine cofactor, which is not part of RNA chain
Comparison of secondary structures of group II self-splicing introns and snRNAs
Spliceosome Spliceosome contains snRNAs, snRNPs and
many other proteins, totally about 300 subunits.
This makes it the most complicted macromolecular machine known to date.
One gene to several proteins
Cleavage at alternative poly(A) sites Alternative promoters Alternative splicing of different exons RNA editing
Alternative splicing, promoters & poly-A cleavage
RNA editing Enzymatic altering of pre-mRNA sequence Common in mitochondria of protozoans and plants and
chloroplasts, where more than 50% of bases can be altered Much rarer in higher eukaryotes
Editing of human apoB pre-mRNA
The two types of editing1) Substitution editing Chemical altering of individual nucleotides Examples: Deamination of C to U or A to I
(inosine, read as G by ribosome)
2) Insertion/deletion editing•Deletion/insertion of nucleotides (mostly uridines) •For this process, special guide RNAs (gRNAs) are required
Guide RNAs (gRNAs) are required for editing
Macromolecular transport across the nuclear envelope
Mechanism for nuclear “import”
Mechanism for nuclear “export”
Mechanism for mRNA transport to cytoplasm
Example of regulation at nuclear transport level: HIV mRNAs
After mRNA reaches the cytoplasm...
mRNA exporter, mRNP proteins, nuclear cap-binding complex and nuclear poly-A binding proteins dissociate from mRNA and gets back to nucleus
5’ cap binds to translation factor eIF4E Cytoplasmic poly-A binding protein (PABPI)
binds to poly-A tail Translation factor eIF4G binds to both eIF4E and
PABPI, thus linking together 5’ and 3’ ends of mRNA
• Polypeptide chain may
be cleaved into two or
three pieces
• Preproinsulin
• Proinsulin - disulfide
bridges
• Insulin
• Secretory protein
Overview Eukaryotic Gene Regulation Models
A. Gene Expression B. Initiation of Transcription C. Posttranscriptional control D. Posttranslational control
•Cleavage
•Post-translational modifications
•Regulatory proteins [products] are activated or
inactivated by the reversible addition of phosphate
groups / phosphorylation
•Sugars on surface of the cell / Glycosylation
5. Control of Translation Block initiation of translation stage
– regulatory proteins attach to 5' end of mRNA • prevent attachment of ribosomal subunits & initiator
tRNA• block translation of mRNA to protein
•Acid/base - act/inact
•Hydrolysis – localization, act/inact
•Acetylation - act/inact
•Phosphorylation - act/inact
•Prenylation - localization
•Glycosylation - targeting
Post-translational modifications
6-7. Protein processing and degradation Protein processing
– folding, cleaving, adding sugar groups, targeting for transport
Protein degradation– ubiquitin tagging– proteasome degradation
•Lifespan of protein is strictly regulated
•Marked protein for destruction is attached by a small protein
ubiquitin
proteasomes
7. Protein degradation
Ubiquitin “Death tag”
– mark unwanted proteins with a label – 76 amino acid polypeptide, ubiquitin– labeled proteins are broken down rapidly in
"waste disposers"proteasomes
1980s | 2004
•Aaron Ciechanover•Israel
•Avram Hershko•Israel
•Irwin Rose•UC Riverside
Proteasome
Protein-degrading “machine”– cell’s waste disposer– breaks down any proteins
into 7-9 amino acid fragments• cellular recycling
The binding of a gene regulatory protein to the major groove of DNA.
Typically, a protein-DNA interface consists of 10 to 20 such contacts, involving different amino acids, each contributing to the binding energy of the protein-DNA interaction.
Zinc finger motif– Common motif in DNA binding, e.g. SP1 has 3
(A) The structure of a fragment of a mouse gene regulatory protein bound to a specific DNA site. This protein recognizes DNA using three zinc fingers of the Cys-Cys-His-His type arranged as direct repeats. (B) The three fingers have similar amino acid sequences and contact the DNA in similar ways. In both (A) and (B) the zinc atom in each finger is represented by a small sphere. (Adapted from N. Pavletich and C. Pabo, Science252:810-817, 1991. © 1991 the AAAS.)
Zinc finger motif
Kadonaga, J.T. 2004. Cell, 116:247.
All of the proteins bind DNA as dimers in which the two copies of the recognition helix (red cylinder) are separated by exactly one turn of the DNA helix (3.4 nm). The second helix of the helix-turn-helix motif is colored blue. The lambda repressor and cro proteins control bacteriophage lambda gene expression, and the tryptophan repressor and the catabolite activator protein (CAP) control the expression of sets of E. coli genes.
Helix-Turn-Helix
Helix-Turn-Helix
Kadonaga, J.T. 2004. Cell, 116:247.
Homeodomains– Related to helix-turn-helix bacterial repressors– Homeobox = 60 AA residues– E.g. en, eve, Hox, Oct-1, Oct-2 (Oct also have Pou domain
next to homeodomain)
The homeodomain is folded into three alfa helices, which are packed tightly together by hydrophobic interactions (A). The part containing helix 2 and 3 closely resembles the helix-turn-helix motif, with the recognition helix (red) making important contacts with the major groove (B). The Asn of helix 3, for example, contacts an adenine. Nucleotide pairs are also contacted in the minor groove by a flexible arm attached to helix 1. The homeodomain shown here is from a yeast gene regulatory protein, but it is nearly identical to two homeodomains from Drosophila, which interact with DNA in a similar fashion. (Adapted from C. Wolberger et al., Cell 67:517-528, 1991. © Cell Press.)
Helix-loop-helix (HLH)
– DNA binding (helix) & dimerization– Class A: ubiquitouslyh expressed proteins, e.g. E12/E47– Class B: tissue-specific expression, e.g. MyoD,
myogenin, Myf-5– Myc proteins (separate class)
Leucine zippers– Dimerization motif– E.g. Jun+Fos = AP1– Gcn4 ->
Helix-loop-helix (HLH)and
Leucine zippers
Kadonaga, J.T. 2004. Cell, 116:247.
Kadonaga, J.T. 2004. Cell, 116:247.
Leucine zippers
Kadonaga, J.T. 2004. Cell, 116:247.
Helix-loop-helix (HLH)
Steroid receptors – Independent domains: DNA binding, hormone
binding, and dimerization
Cortisol - glucocorticoid receptor (GR).
Retinoic acid - retinoic acid A receptor (RAR).
Thyroxine - thyroid hormone receptor (TR).
Figure 1 Genome-wide comparison of transcriptional activator families in eukaryotes. The relative sizes of transcriptional activator families among Homo sapiens, D. melanogaster, C. elegans and S. cerevisiae are indicated, derived from an analysis of eukaryotic proteomes using the INTERPRO database, which incorporates Pfam, PRINTS and Prosite. The transcription factors families shown are the largest of their category out of the 1,502 human protein families listed by the IPI.
Posttranslational ModificationModification Charge-dependent change
Acylation loss of a-amino positive charge
Alkylation alteration of a- or e-amino positive group
Carboxylmethylation esterification of specific carboxyl group
Phoshorylation mainly modify Ser, Thr and Tyr
Sulfation mainly modify Tyr
Carboxylation bring negative charge
Sialyation mainly on Asn, Thr and Ser
Proteolytic processing truncation leads to change of pI
Posttranslational Modification
Location Modification Nucleus acetylation, phosphorylationLysosome mannose-6-phosphate labelled N-linked sugarMitochondria N-formyl acylationGolgi N- and O-linked ologosaccharide, sulfation, palimitoylationER N-linked oligosaccharide, GPI-anchorCytosol acetylation, methylation, phosphorylation, Ribosome myristoylationPlasma membrane N- and O-glycosylation, GPI-anchorExtraceullar fluid N- and O-glycosylation, acetylation, phosphorylation Extrallular matrix N- and O-glycosylation, phosphorylation, hydroxylation
Posttranslational Modification
Examples:
Chromatin Structure/function - acetylation Regulation of mitochondrial processes –
phosphorylation Evade immune system – glycosylation Gene regulation – glycosylation Recognition - glycosylation
Chromatin Remodeling – mechanisms for transcription-associated structural changes in chromatin
•• transcription•• post transcription (RNA stability)
•• post transcription (translational control)•• post translation (not considered gene regulation)
usually, when we speak of gene regulation, we are referring to transcriptional regulation
the “transcriptome”
Genes can be regulated at many levels
RNA PROTEINDNATRANSCRIPTION TRANSLATION
The “Central Dogma”
Gene expression must be regulated in:
•TIME
• Wolpert, L. (2002) Principles of Development New York: Oxford University Press. p. 31
•SPACE
• Paddock S.W. (2001). BioTechniques 30: 756 - 761.
Gene expression must be regulated in:
•ABUNDANCE
•Stern, D. (1998). Nature 396, 463 - 466
Gene expression must be regulated in:
What happens when gene regulation goes awry?
Disease - chronic myeloid leukemia - rheumatoid arthritis
•1
•2•3 •4 •5 •6
Developmental abnormalities (birth defects)
•photo credits: Wolpert, L. (2002) Principles of Development New York: Oxford University Press. pp. 183, 340
Summary of Regulation of Gene Expression
(summary of the report)
initiation of transcription
1
mRNA splicing
2
mRNA protection3
initiation of translation
6
mRNAprocessing
5
1 & 2. transcription - DNA packing - transcription
factors3 & 4. post-transcription
- mRNA processing
- splicing- 5’ cap & poly-A tail- breakdown by siRNA
5. translation- block start of
translation6 & 7. post-translation - protein
processing- protein degradation
7 protein processing & degradation
4
4
Gene Regulation
Conclusion of Regulation of Gene Expression
Regulation at transcriptional level: Regulation of initiation of transcription
Chromatin-mediated transcriptional control Activators and repressors interaction with transcription complex
Regulation at post-transcriptional level in the nucleus: Regulation of alternative splicing leading to production of multiple
isoforms of proteins Regulation of transport of mRNA into cytoplasm
Regulation at post-translational level in cytoplasm Micro RNAs RNA intereference (RNAi or siRNA) Cytoplasmic polyadenylation mRNA degradation Localization of mRNA in the cytoplasm
Sources B Lewin, Genes VII Lodish et al., Molecular Cell Biology EH Davidson: Genomic Regulatory Systems Alberts et al., Essential Cell Biology Blackwood, E.M. & J.T. Kadonaga: Going the
distance: a current view of enhancer action. Cell, February 22, 2002: 108 (4) "Reviews on
Gene Expression"
Sources (cont…) Campbell, N.A., J.B. Reece, M.L. Cain, R.B.
Jackson & P.V. Minorsky. 1996-2010. Biology, Benjamin-Cummings Publishing Company.
Kadonaga, J.T. 2004. Transcription in eukaryote. Cell, 116:247.
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NOE P. MENDEZMaster of Science in Biology
Central Mindanao [email protected]