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Eukaryotic gene regulation models (by np mendez)
1. Prepared by:Prepared by:
NOE P. MENDEZNOE P. MENDEZ
Master of Science in BiologyMaster of Science in Biology
CENTRALMINDANAO UNIVERSITYCENTRALMINDANAO UNIVERSITY
EUKARYOTIC GENE
REGULATION MODELS
2. Overview
Eukaryotic Gene Regulation Models
A. Gene Expression
B. Initiation of Transcription
C. Posttranscriptional control
D. Posttranslational control
3. What is a gene?
“The entire nucleic acid sequence that is
necessary for the synthesis of a functional
polypeptide or RNA molecule.”
7. 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.
8. 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
9. 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.
15. Overview
Eukaryotic Gene Regulation Models
A. Gene Expression
B. Initiation of Transcription
C. Posttranscriptional control
D. Posttranslational control
16. • chromatin changes
• transcription
• processing RNA
• transport to cytoplasm
• degradation of mRNA
• translation
• cleavage, chemical modification
• protein degradation
Complicated regulation system
17. Binding may form the biochemical basis of translational
synergy between cap structure and poly (A) tail
(Preiss & Hentze, 1999).
19. •Signal
•NUCLEUS
•Chromatin
•Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
•DNA
•Gene
•Gene available
for transcription
•RNA •Exon
•Primary transcript
•Transcription
•Intron
•RNA processing
•Cap
•Tail
•mRNA in nucleus
•Transport to cytoplasm
•CYTOPLASM
•mRNA in cytoplasm
•Translation•Degradation
of mRNA
•Polypeptide
•Protein processing, such
as cleavage and
chemical modification
•Active protein
•Degradation
of protein
•Transport to cellular
destination
•Cellular function (such
as enzymatic activity,
structural support)
21. 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
22. 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
23. 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
24. • 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
25. 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
26. 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
28. 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
42. Overview
Eukaryotic Gene Regulation Models
A. Gene Expression
B. Initiation of Transcription
C. Posttranscriptional control
D. Posttranslational control
43. 3. Processing RNA
•Post-transcriptional modifications
•Alternative RNA splicing
•The same primary transcript, but different
the mRNA molecule / exons and introns
44. 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
46. 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
47. Action of siRNA
siRNA
double-stranded
miRNA + siRNA
mRNA degraded
functionally turns
gene off
Hotnew topicin biology
mRNA for translation
breakdown
enzyme
(RISC)
dicer
enzyme
49. 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.
50. Capping mechanism in mammals
DNA
Growing
RNA
Capping enzyme is allosterically controlled by
CTD domains of RNA Pol II and another
stimulatory factor hSpt5
58. p
p
Pol II
c
t
d
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
p
p
aataaa
FCP1 Phosphatase
removes phospates
from CTDs
cap
63. 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
64. Binding of U1 and
U2 snRNPs
Binding of U4,
U5 and U6
snRNPs
The essential steps in splicing
68. 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
70. 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
71. Group I introns utilize guanosine cofactor, which is not part of RNA
chain
73. Spliceosome
Spliceosome contains snRNAs, snRNPs and
many other proteins, totally about 300
subunits.
This makes it the most complicted
macromolecular machine known to date.
74. One gene to several proteins
Cleavage at alternative poly(A) sites
Alternative promoters
Alternative splicing of different exons
RNA editing
76. 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
77. The two types of editing
1) 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
84. 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
85.
86.
87. • Polypeptide chain may
be cleaved into two or
three pieces
• Preproinsulin
• Proinsulin - disulfide
bridges
• Insulin
• Secretory protein
88. Overview
Eukaryotic Gene Regulation Models
A. Gene Expression
B. Initiation of Transcription
C. Posttranscriptional control
D. Posttranslational control
89. •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
91. 6-7. Protein processing and degradation
Protein processing
– folding, cleaving, adding sugar groups,
targeting for transport
Protein degradation
– ubiquitin tagging
– proteasome degradation
92. •Lifespan of protein is strictly regulated
•Marked protein for destruction is attached by a small protein
ubiquitin
proteasomes
7. Protein degradation
93. 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
95. 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.
99. 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
101. 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.
102. 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 ->
107. 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.
108. Posttranslational Modification
Modification 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
111. Chromatin Remodeling – mechanisms for
transcription-associated structural changes in chromatin
112. •• 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 PROTEINDNA
TRANSCRIPTION TRANSLATION
The “Central Dogma”
113. Gene expression must be regulated in:
•TIME
• Wolpert, L. (2002) Principles of Development New York: Oxford University Press. p. 31
114. •SPAC
E
• Paddock S.W. (2001). BioTechniques 30: 756 - 761.
Gene expression must be regulated in:
120. initiation of
transcription
1
mRNA splicing
2
mRNA
protection
3
initiation of
translation
6
mRNA
processing
5
1 & 2. transcription
- DNA packing
- transcription
factors
3 & 4. post-transcription
- mRNA
processing
- splicing
- 5’ cap & poly-A
tail
- breakdown by
siRNA
5. translation
- block start of
translation
6 & 7. post-translation
- protein
processing
- protein
degradation
7 protein
processing &
degradation
4
4
Gene Regulation
121.
122.
123.
124.
125.
126.
127.
128.
129. 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
130. 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"
DEFINITION OF TERMS
Coactivator
– binds to and affects activator protein which binds to DNA.
– Does not itself bind to DNA.
– Adapter molecules that integrate signals from activators and perhaps repressor and relay the results to basal factors
Corepressors
– binds to and affects silencer/repressor protein which binds to DNA
– Does not itself bind to DNA.
Enhancers
– sequence of DNA that increases the activity of the promoter.
Silencers
– sequence which decreases the activity of the promoter.
Activators
– proteins that binds at enhancers.
Repressors
– proteins that bind to silencers.
Transcription-control regions
Signals for 3’ cleavage and polyadenylation
Signals for splicing of primary RNA transcripts
Mutations in these signals prevent expression of a functional mRNA and thus of the encoded protein
Exons a segment of a DNA or RNA molecule containing information coding for a protein or peptide sequence.
FIGURE 2: General pattern of control elements that
regulate gene expression in multicellular eukaryotes and
yeast. (a) Genes of multicellular organisms contain both
promoter-proximal elements and enhancers, as well as a TATA
box or other promoter element. The promoter elements position
RNA polymerase II to initiate transcription at the start site and
influence the rate of transcription. Enhancers may be either
upstream or downstream and as far away as 50 kb from the
transcription start site. In some cases, enhancers lie within
introns. For some genes, promoter-proximal elements occur
downstream from the start site as well as upstream. (b) Most
S. cerevisiae genes contain only one regulatory region, called an
upstream activating sequence (UAS), and a TATA box, which is
≈90 base pairs upstream from the start site.
RNA pol II is located in the nucleoplasm and is responsible for transcription
of the vast majority of genes including those encoding mRNA, small nucleolar RNAs (snoRNAs), some
small nuclear RNAs (snRNAs), and microRNAs. Gene transcription is a remarkably complex process. The
synthesis of tens of thousands of different eukaryotic mRNAs is carried out by RNA pol II. During the
process of transcription, RNA pol II associates transiently not only with the template DNA but with many
different proteins, including general transcription factors.
Figure 3: Comparison of a simple and complex RNA pol II transcription unit. (A) A typical yeast
(unicellular eukaryote) transcription unit. The start of transcription (+1) of the protein-coding gene (transcription unit)
is indicated by an arrow. (B) A typical mammalian (multicellular eukaryote) transcription unit with clusters of proximal
Promoter elements and long-range regulatory elements located upstream from the core promoter (TATA). There is
variation in whether a particular element is present or absent, the number of distinct elements, their orientation relative
to the transcriptional start site, and the distance between them. Although the figure is drawn as a straight line, the
binding of transcription factors to each other draws the regulatory DNA sequences into a loop (Kadonaga, 2004).
Insulators contain clustered binding sites for sequence-specific DNA-binding proteins. The
exact molecular mechanism by which they block enhancers and silencers is not clear. One model proposes
that insulators tether the DNA to subnuclear sites, forming loops that separate the promoter of one gene
from the enhancer of another (Kadonaga, 2004).
Insulators are
stretches of DNA (as few as 42 base pairs may do the trick)
located between the
o enhancer(s) and promoter or
o silencer(s) and promoter of adjacent genes or clusters of adjacent genes.
Their function is to prevent a gene from being influenced by the activation (or repression) of its neighbors.
Transcription Factor IIB (TFIIB) - binds both the DNA and pol II.
In molecular biology and genetics, upstream and downstream both
refer to a relative in DNA or RNA. Each strand of DNA or RNA has a 5'
end and a 3' end, so named for the carbon position on the deoxyribose
(or ribose) ring. ...Upstream is toward the 5' end of the RNA molecule
and downstream is toward the 3' end.
In an RNA, anything towards the 5' end of a reference point is "upstream"
of that point. This orientation reflects the direction of both the synthesis of
mRNA, and its translation - from the 5' end to the 3' end. In DNA, the situation
is a bit more complicated. In the vicinity of a gene(or in a cDNA), the DNA
has two strands, but one strand is virtually a duplicate of the RNA, so it's 5'
and 3' ends determine upstream and downstream, respectively. NOTE that
in genomic DNA, two adjacent genes may be on different strands and thus
oriented in opposite directions. upstream or downstream is only used on
conjunction with a given gene.
THE MOLECULAR MECHANISMS THAT CREATE SPECIALIZED CELL TYPES
All cells must be able to switch genes on and off in response to signals in
their environments. But the cells of multicellular organisms have evolved
this capacity to an extreme degree and in highly specialized ways to form
an organized array of differentiated cell types. In particular, once a cell in a
multicellular organism becomes committed to differentiate into a specific
cell type, the choice of fate is generally maintained through many subsequent
cell generations. This means that the changes in gene expression,
which are often triggered by a transient signal, must be remembered. This
phenomenon of cell memory is a prerequisite for the creation of organized
tissues and for the maintenance of stably differentiated cell types.
In contrast, the simplest changes in gene expression in both eukaryotes
and bacteria are often only transient; the tryptophan repressor, for example,
switches off the tryptophan genes in bacteria only in the presence of
tryptophan; as soon as the amino acid is removed from the medium, the
genes are switched back on, and the descendants of the cell will have no
memory that their ancestors had been exposed to tryptophan.
In this section, it discusses some of the special features of transcriptional
regulation that are found in multicellular organisms. The focus will be
on how these mechanisms create and maintain the specialized cell types
that give a worm, a fly, or a human its distinctive characteristics (Kadonaga, 2004).
Gene expression evolved to maintain constant internal conditions while facing changing external conditions.
Figure 8–2 Differentiated cells contain all
the genetic instructions necessary to direct
the formation of a complete organism.
(A) The nucleus of a skin cell from an adult
frog transplanted into an egg whose nucleus
has been removed can give rise to an entire
tadpole. The broken arrow indicates that to
give the transplanted genome time to adjust
to an embryonic environment, a further
transfer step is required in which one of the
nuclei is taken from the early embryo that
begins to develop and is put back into a
second enucleated egg. (B) In many types of
plants, differentiated cells retain the ability
to “dedifferentiate,” so that a single cell can
form a clone of progeny cells that later give
rise to an entire plant. (C) A differentiated
cell from an adult cow introduced into an
enucleated egg from a different cow can
give rise to a calf. Different calves produced
from the same differentiated cell donor are
genetically identical and are therefore
clones of one another (Gurdon, 1968).
An operon is a set of genes and the switches that control the expression of those genes.
An operon consists of:
- operator
- promotor
- and genes that they control
All together, the operator, the promoter and the genes they control – the entire stretch of DNA required for enzyme production for the pathway – is called an operon.
Figure 8–3 Eukaryotic gene expression
can be controlled at several different
steps. Examples of regulation at each of
the steps are known, although for most
genes, the main site of control is step 1:
transcription of a DNA sequence into RNA.
Red box: Transcriptional control
Green boxes: Posttranscriptional control
Purple box: Translational control
Dark blue box: Posttranslational control
The initiation of transcription occurs in transcriptional control.
Figure 18.6 Stages in gene expression that can be regulated in eukaryotic cells.
Packing of promoter DNA into nucleosomes affects Initiation of Transcription
Initiation of transcription in eukaryotic cells must also take into account the packaging of DNA into chromosomes.
The genetic material in eukaryotic cells is packed into nucleosomes, which, in turn, are folded into higher-order
structures. How do transcription regulators, general transcription factors, and RNA polymerase gain access to such
DNA? Nucleosomes can inhibit the initiation of transcription if they are positioned over a promoter, probably because
they physically block the assembly of the general transcription factors or RNA polymerase on the promoter. In fact,
such chromatin packaging may have evolved in part to prevent leaky gene expression—initiation of transcription in the
absence of the proper activator proteins. In eucaryotic cells, activator and repressor proteins exploit chromatin
structure to help turn genes on and off. Chromatin structure can be altered by chromatin-remodeling complexes and
by enzymes that covalently modify the histone proteins that form the core of the nucleosome. Many gene activators
take advantage of these mechanisms by recruiting these proteins to promoters. For example, many transcription
activators attract histone acetylases, which attach an acetyl group to selected lysines in the tail of histone proteins.
This modification alters chromatin structure, probably allowing greater accessibility to the underlying DNA; moreover,
the acetyl groups themselves are recognized by proteins that promote transcription, including some of the general
transcription factors.
"At actual size, a human cell's DNA totals about 3 meters in length." McGraw Hill Encyclopedia of Science and Technology. New York: McGraw Hill, 1997.
"If stretched out, would form very thin thread, about 6 feet (2 meters) long."
Or let’s say Regulation of Chromatin Structure
Constitutive Heterochromatin: always inactive and condensed: e.g. repetitive DNA, centromeric DNA
Facultative Heterochromatin: can exist in both forms. E.g.: Female X chromosome in mammals.
Figure 18.7 A simple model of histone tails and the effect of histone acetylation.
-Chromatin-modifying enzymes provide initial control of gene expression by
making a region of DNA either more or less able to bind the transcription machinery.
-Once the chromatin of a gene is optimally modified for expression, transcription
is the next major step where gene expression is regulated.
-Associated with most eukaryotic genes are multiple control elements, segments
of noncoding DNA that serve as binding sites for transcription factors that help regulate
Transcription.
-Control elements and the transcription factors they bind are critical to the precise regulation
of gene expression in different cell types.
-Some transcription factors function as repressors, inhibiting expression of a particular gene by a variety
of methods.
-Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription.
The Roles of Transcription Factors
-To initiate transcription, eukaryotic RNA polymerase requires the assistance of
proteins called transcription factors.
-General transcription factors are essential for the transcription of all
protein-coding genes.
-In eukaryotes, high levels of transcription of particular genes depend on
control elements interacting with specific transcription factors.
Figure 18.8 A eukaryotic gene and its transcript.
-Proximal control elements are located close to the promoter.
-Distal control elements, groupings of which are called enhancers,
may be far away from a gene or even located in an intron.
-The functioning of enhancers is an example of transcriptional
control of gene expression.
-In Eukaryotes, the rate of gene expression can be strongly increased
or decreased by the binding of specific transcription factors either
activators or repressors
Figure 18.8 A eukaryotic gene and its transcript.
Posttranscriptional modification of RNA
- (removing introns and connecting exons)
Figure 18.8 A eukaryotic gene and its transcript.
Figure 18.9 The structure of MyoD, a specific transcription factor that acts as an activator.
RNA polymerase II (pol II, also known as RNAP II) is a complex of 12 different proteins.
TATA-binding protein (TBP)
- recognizes and binds to the TATA box .
- 14 other protein factors which bind to TBP and each other, but not to the DNA.
In eukaryotes, gene activation occurs at a distance.
An activator protein bound to DNA attracts RNA polymerase
and general transcription factors to the promoter. Looping of
the DNA permits contact between the activator protein bound
to the enhancer and the transcription complex bound to the
promoter. In the case shown here, a large protein complex
called Mediator serves as a go-between. The broken stretch
of DNA signifies that the length of DNA between the enhancer
and the start of transcription varies, sometimes reaching tens
of thousands of nucleotide pairs in length.
Figure 18.10 A model for the action of enhancers and transcription activators.
Figure 18.10 A model for the action of enhancers and transcription activators.
Figure 18.10 A model for the action of enhancers and transcription activators.
Processing of eukaryotic pre-mRNA
About 60% of human genes give spliced mRNAs
Eukaryotic cells have evolved RNA surveillance mechanisms that prevent incorrectly processed RNAs to be transported out of the nucleus
Processing of eukaryotic pre-mRNA
-capping
-polyadenylation
-splicing
-editing
Nuclear transport
YSPTSPS
Poly A site
– 10-35 downstream aauaaa, g/u – 50 downstream polyA site
Mutations in ESEs can cause exon skipping. Spinal muscle atrophy – genetic cause of childhood mortality
apob- apolipoprotein B in humans
– part of large lipopr. complexes that transport lipids to serum.
- Only apob100 containing compl. transport cholesterol to body
tissues via low density lipopr. complexes by binding to LDL receptors
gap – gtpase accelerating protein ran- g protein in 2 different conformations ran gets back in nucleus by aid of nuclear transport factor 2 ntf2
nes- leucine rich nuclear export signal
mRNA exporter: big sub: 3 domains, m and c bind to fg repeats, m also to small sub
mRNP proteins – SRs,
Ubiquitination requires 3 enzymes:
- E1 (ubiquitin-activating enzyme) activates ubiquitin (U)
- E2 (ubiquitin-conjugating enzyme) acquires U via high-energy thioester
- E3 (ubiquitin ligase) transfers U to target proteins
- Hierarchical organization: one or few E1s exist, more E2s, many E3s.
Usually, the addition of polyubiquitin chains targets a protein for degradation by the proteasome.
However, the addition of one ubiquitin (monoubiquitinylation) can alter the function of a protein
without signaling its destruction. A conjugating enzyme catalyzes the formation of a peptide bond
between ubiquitin and the side chain-NH2 of a lysine residue in a target protein. For example,
monoubiquitinylation of histone H2B is, depending on the gene, associated with activation or silencing
of transcription as well as transcription elongation. Monoubiquitinylation of linker histone H1 leads to its
release from the DNA. In the absence of the linker histone, chromatin becomes less condensed, leading
to gene activation.
The Proteasome - high molecular weight (28S) protease complex that degraded
ubiquitinated proteins in the cytoplasm
- Present in cytoplasm and nucleus, not ER
- Uses ATP
- Contains a 700 kD protease core and two 900 kD regulatory domains.
- Highly conserved and similar to proteases found in bacteria.
- Shaped like a cylinder.
- Proteins enter the cavity, and are cleaved into small peptides.
- Most but not all proteasome substrates are ubiqutinated.
Figure 8–4 A transcription regulator binds
to the major groove of a DNA helix. Only
a single contact between the protein and one
base pair in DNA is shown. Typically, the
protein–DNA interface would consist of 10–20
such contacts, each involving a different amino
acid and each contributing to the strength of
the protein–DNA interaction.
The protein forms hydrogen bonds, ionic bonds,
and hydrophobic interactions with the edges of
the bases, usually without disrupting the hydrogen
bonds that hold the base pairs together. Although
each individual contact is weak, the 20 or so
contacts that are typically formed at the protein–DNA
interface combine to ensure that the interaction is
both highly specific and very strong; indeed, protein–DNA
interactions are among the tightest and most specific
molecular interactions known in biology.
Figure 8–4 A transcription regulator binds
to the major groove of a DNA helix. Only
a single contact between the protein and
one base pair in DNA is shown. Typically,
the protein–DNA interface would consist
of 10–20 such contacts, each involving a
different amino acid and each contributing
to the strength of the protein–DNA
interaction.
The protein forms hydrogen bonds,
ionic bonds, and hydrophobic interactions with the edges of the bases,
usually without disrupting the hydrogen bonds that hold the base pairs
together (Figure 8–4). Although each individual contact is weak, the 20 or
so contacts that are typically formed at the protein–DNA interface combine
to ensure that the interaction is both highly specific and very strong;
indeed, protein–DNA interactions are among the tightest and most specific
molecular interactions known in biology.
Figure 11.17 The basic DNA-binding domain. (A) The leucine zipper
and the basic DNA-binding domain are illustrated by the transcription factor
AP-1–DNA complex. AP-1 is a dimer formed by Jun and Fos. Two helices
“zipper” together by their leucine residues (inset) (Protein Data Bank, PDB: 1FOS.)
(B) (Left) Schematic representation of the basic helix-loop-helix motif and the
basic DNA-binding domain. (Right) Three-dimensional image of a Myc–Max–DNA
complex. (Protein Data Bank, PDB: 1NKP).
Basic leucine zipper (bZIP)
The bZIP motif is not as common as the HTH or zinc finger motifs. The motif was first described in 1987
for the CAAT/enhancer-binding protein (C/EBP), which recognizes both the CCAAT box found in many
viral and mammalian gene promoters and the “core homology” sequence common to many enhancers.
The structure of the DNA-binding domain was solved by comparison with two other related proteins, the
GCN4 regulatory protein from yeast and the transcription factor Jun. The latter protein is encoded by the
proto-oncogene c-jun. Proto-oncogenes are genes that have a normal function in the cell but can become
cancer-causing when they undergo certain mutations.
The bZIP motif is not itself the DNA-binding domain of the transcription factor and does not directly
contact the DNA. Instead, it plays an indirect structural role in DNA binding by facilitating dimerization
of two similar transcription factors. The joining of two bZIP-containing transcription factors results in the
correct positioning of the two adjacent DNA-binding domains in the dimeric complex. The
bZIP motif is a stretch of amino acids that folds into a long α-helix with leucines in every seventh position.
The leucines form a hydrophobic “stripe” on the face of the α-helix. Two polypeptide chains with this
motif coil around each other to form a dimer in a “coiled coil” arrangement. The amino acids protrude like
knobs from one α-helix and fit into complementary holes between the knobs on the partner helix, like a
zipper. The dimer forms a Y-shaped structure and one end of each α-helix protrudes into the major groove
of the DNA. This allows the two basic binding regions (rich in arginine and lysine) to contact the DNA.
The dimer can be either a homodimer – two of the same polypeptide – or a heterodimer – two different
polypeptides zipped together. For example, C/EBP forms both homodimers and heterodimers composed
of mixed pairs of different variants. The transcription factor AP-1 is a combination of members of two
different families of transcription factors, Fos and Jun. Tony Kouzarides and Edward Ziff showed that at
low concentrations of protein Jun and Fos bind to their DNA target better together than either one does
separately. They also demonstrated that the bZIP domains of each protein are essential for
binding. Jun can form both homodimers and heterodimers, whereas Fos can only form heterodimers.
Basic helix-loop-helix (BHLH)
The BHLH motif is distinct from the HTH motif (Fig. 11.17B). It forms two amphipathic helices,
containing all the charged amino acids on one side of the helix, which are separated by a nonhelical
loop. Like the bZIP motif, the BHLH motif is not itself the DNA-binding domain of the transcription
factor and does not directly contact the DNA. Instead, it plays an indirect structural role in DNA
binding by facilitating dimerization of two similar transcription factors. The joining of two BHLH
containing transcription factors results in the correct positioning of the two adjacent DNA-binding
domains in the dimer. The DNA-binding domains are rich in basic amino acids and can interact directly
with the acidic DNA. As an example, efficient DNA binding by the mouse transcription factor Max
requires dimerization with another BHLH protein. Max binds DNA as a heterodimer with either with
Myc or Mad. The Myc–Max complex is a transcriptional activator, whereas the Mad–Max complex is
a repressor.
