Eukaryotic gene regulation models (by np mendez)

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:

• 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


elements)•DNA


•Proximalcontrol



•Poly-Asignal


terminationregion

•Downstream•Poly-Asignal

•Exon •Intron •Exon •Exon•Intron

•Transcription

•Cleaved3 end ofprimary

transcript

•5•Primary RNA

transcript(pre-mRNA)


elements)•DNA


•Proximalcontrol



•Poly-Asignal


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


PromoterGene

TATA boxGeneral

transcriptionfactors

DNA-bendingprotein

Group of mediator proteins

ActivatorsDNA


PromoterGene

TATA boxGeneral

transcriptionfactors

DNA-bendingprotein

Group of mediator proteins

RNApolymerase II

RNApolymerase II

RNA synthesisTranscription

initiation complex

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

•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


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



Leucine zippers


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.


•ABUNDANCE

•Stern, D. (1998). Nature 396, 463 - 466


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.

Sources (cont…)

•Turn yourQuestion Genes on!

THANK YOU FOR

LISTENING!

NOE P. MENDEZMaster of Science in Biology

Central Mindanao [email protected]

Eukaryotic gene regulation models (by np mendez)

Science

Transcript of Eukaryotic gene regulation models (by np mendez)