Transcript Protein Biosynthesis at Three Levels of Modifications

In vivo
Protein
Modification
Two Modes of Protein Modifications:
1. Reversible Reactions (on-off switch)
1. Phosphorylation of Ser/Thr/Tyr
2. Adenylation of Tyr
3. ADP-ribosylation of Arg
4. Methylation of the COOH groups
5. O-Glycosylation
6. Palmitoylation of Cys residues
Reversible reactions can be regarded as the control of cellular activities.
2. Non-reversible reactions
1. Spontaneous reactions: Gln → Glu or Asn → Asp, Glycosylation of α- or ε-NH2 groups
2. Cross-links: Cys-Cys cross-linking in stabilizing protein structure, γGlu-εLys cross-linking in
fibrin clots, -S-S- cross-linking, Alanino-His in hard tissue proteins, His-Cys thioether, Ser(Cys)γGlu esters
3. Covalently attached cofactors: Biotin to Lys, Heme to Cys, Pantothenyl phosphate to Ser, Flavin
to Tyr, Cys or His
4. Membrane anchors: Myristate to Gly, Glycosyl Phosphatidyl Inositol to COOH-terminal,
Isoprenyl to Cys
5. Ubiquitination for protein degradation
6. N-Glycosylation for many cellular functions
Classical Protein Biosynthesis
1. Proteins are synthesized in ribosomes and one
trinucleotide specifies one amino acid.
2. Codons are universal and the starting codon (AUG)
specifies Met or fMet.
3. Every protein should start with Met or fMet at the NH2terminus.
4. Every protein should have no more than 20 amino acids.
However, many exceptional amino acids were found in
many naturally occurring proteins, therefore, proteins must
be modified before or after ribosomal protein synthesis.
Protein Biosynthesis at Three Levels of
Modifications
20 Amino acids ＋ 20 tRNA’s
Pre-translational
Modifications
20 aa-tRNA’s
Co-translational
Modifications
Nascent polypeptide
Post-translational
Modifications
Completed polypeptide
↓
↓
↓
Examples of Three Levels of Protein Modifications
Levels
1. Pre-translational
Examples
a) Selenocysteine t-RNA
b) Nonnatural amino acid t-RNA
2. Co-translational a) Signal sequence cleavage
b) N-Glycosylation
3. Post-translational a) Glucosylation and O-Glycosylation
b) Peptide bond cleavage
c) Protein splicing
d) Lipidation
e) Disulfide bond formation
f) Ubiquitination
Selenocystyl Proteins
Biosynthesis of Noncanonical Aminoacyl-tRNA:
Selenocystyl-tRNA
- H2O
tRNASec ＋ Serine
Ser-tRNASec
Aminoacrylyl-tRNASec
+Selenophosphate
Selenocysteyl-tRNASec
(ATP ＋ Se2-
Selenophosphate ＋ AMP ＋ Pi)
Incorporation of Selenocysteine into Proteins
Gene sequences around the in-frame TGA codon for selenocysteine
are normally coded for the stop codon. In the absence of selenium,
the Se-Cys-containing proteins terminate at this codon.
Proteins Containing Selenocysteine
Designers Proteins
Properties of Aminoacyl-tRNA Synthetases (AARSs)
The 3’-terminal A is
aminoacylated by the AARSs
and the 20 amino acids are
specifically joined to their
cognate (同族) tRNAs.
Thus, the aminoacyl-tRNAs (AA-tRNAs) are the main experimental
efforts to incorporate nonnatural amino acids into proteins.
Changing Relationships Between an Amino Acid and a
tRNA
1. The concept of tRNA identity provided recognition of cognate tRNAs
between species. This led to expression of heterologous AARSs in vivo
as a means to incorporate the wrong amino acid into a protein at a
specific position.
2. A heterologous synthetase-tRNA pair could be introduced into an
organism and operate as an extra pair that was “orthogonal” (正規) to the
existing homologous set of 20 AARSs.
3. Orthogonality means that the new AARS does not mischarge any of the
tRNAs from the host organism and the new tRNA is not a substrate for
any host AARS.
4. Thus, an organism engineered has 21 noncross-reacting AARSs that can
be further manipulated so that the new twenty-first pair brings in a novel
amino acid.
Use of Amino Acid Auxotrophs
1. Incorporation of noncanonical (非典型) amino acids into proteins
was discovered from the study of amino acid analogs synthesized by
plants. These amino acids are toxic to microorganisms because they
are misincorporated into proteins in place of a related canonical
amino acid.
2. In strains auxotrophic for an encoded amino acid, high levels of
substitution by an analog could be achieved. These analogs are only
bacteriostatic and not bactericidal.
3. Thus, misincorporation of canavanine leads to cell death, but
selenomethionine or trifluoroleucine which are well tolerated.
4. The main mechanism for analogs to be incorporated into proteins is
provided by AARSs. Thus, bypassing the synthetase’s specificity,
and mischarging of an analog onto a tRNA can lead to insertion of
the analog into a growing polypeptide chain.
Selection for Replacement of an Amino Acid by
an Analog
1. Selection methods have also been used to replace a canonical
amino acid with an analog.
2. Cells can replace Leu with trifluoro-Leu with epigenetic
adaptation. However, a low percentage ( 5%) of the natural
amino acid was still present.
3. Total replacement of Trp with 4-fluoro-Trp could be achieved
with a small number of genome-wide mutations in B. subtilis.
Multisite Misincorporation in
Overexpressed Proteins
1. Most amino acid analogs are too toxic to promote sustained
exponential growth. However, modified proteins can be
overexpressed in nondividing cells if enough biomass has
been generated prior to induction.
2. By washing cells and replacing the exogenously added
canonical amino acid with its analog just before inducing
gene expression, high levels of misincorporation (80% to 99%)
of nonnatural analogs into target proteins can be achieved,
with good yields of the purified proteins (10–100 mg/L).
Multi-site Incorporation of Analogs into E. coli Proteins
Analog
Target AARS
Perthiaproline
Norleucine
ProRS
MetRS
Selenomethionine
4-Fluorotryptophan
p-Fluorophenylalanine
β-(Thienopyrrolyl)alanines
Aminotryptophans
2-Methylhistidine
3-Fluorotyrosine
O-Methylthreonine
3,4-Dehydroproline
MetRS
TrpRS
PheRS
TrpRS
TrpRS
HisRS
TyrRS
IleRS
ProRS
Yields
38%
100%
100%
75%
60%
Applications
Drug carrier
Increased enzyme
activity
Crystallography
NMR
Tracer
Chromophore
PH sensors
Expanding the Genetic Code:
Use of the Stop Codon for Coding Unnatural Amino Acids
1. The unnatural amino acid is added to the cell growth medium, taken
up by the cell.
2. It is specifically recognized by an ‘orthogonal’ aminoacyl-tRNA
synthetase and attached to the orthogonal amber suppressor tRNA.
3. It is then decoded on the ribosome in response to an introduced
amber codon (UAG), allowing its incorporation into the peptide.
Selection of a New Orthogonal Synthetase-tRNA Pair
A heterologous aminoacyl-tRNA synthetase–tRNA pair is imported
into a host containing a set of natural synthetases and the subsequent
selection of a mutated active site in the new orthogonal synthetase that
recognizes an unnatural amino acid.
Positive and Negative Selection Procedure
1. To generate a synthetase with this altered specificity, a large library of active-site
variants of the synthetase is subject to positive selection for activity with either natural or
unnatural amino acids, by virtue of their ability to suppress an introduced stop codon and
so allow complete translation of a gene that is essential for survival.
2. The synthetases that use natural amino acids are subsequently removed by a negativeselection step, in which they use natural amino acids to suppress a stop codon introduced
in a toxic gene, which leads to cell death.
Application I:
Site-Specific Modification at 53 Position of Sec Y
A photocrosslinking reagent, p-benzoyl-l-phenylalanine (Bpa),
provides a powerful tool to gain information about the
interaction of a specific protein with another molecule.
To analyse the basis of the interaction
between SecA and SecY, Bpa was
introduced at 53 positions in the
cytoplasmic loops of SecY.
The protein was then crosslinked in
response to the light, forming covalent
adducts with SecA.
Covalently linked SecA–SecY complexes
can then be isolated through cell lysis, and
the complexes can be detected by SDS–
PAGE and western blot analysis with
antibodies against SecA.
Application II:
Genetically Encoded Post-Translational Modifications
Genetically encoded Nε-Lys acetylation allows the role of
acetylation in DNA ‘breathing’ to be assessed by single-molecule
fluorescence resonance energy transfer (FRET).
When FRET is measured
between a donor and an acceptor
fluorophore on DNA, a larger
fraction of nucleosomes
containing acetylated Lys56 on
histone H3 are found with low
FRET efficiency, suggesting that
Lys56 acetylation favours DNA
breathing and a more open
conformation.
Pharmaceutical Applications:
The HIV Protease Inhibitors
1. Modified peptides are key pharmaceuticals for the treatment
of diseases. A prominent class of compounds in this category
are the protease inhibitors.
2. The HIV protease inhibitors, containing a nonhydrolyzable
peptide backbone at the site of proteolysis, are synthesized
through incorporation of nonnatural amino acid.
Biophysical Study Applications
1. The replacement of Met by Se-Met has been extensively used for phase
determination in protein structure studies.
2. A spin-labeled nonnatural amino acid, containing nitroxyl 1-oxyl2,2,5,5-tetramethylpyrroline, was site-specifically incorporated into a T4
lysozyme using in vitro translation to yield a protein that had an electron
paramagnetic resonance signal.
3. An E. coli strain that was auxotrophic for tryptophan was grown in the
presence of 4-aminotryptophan. Incorporation of this amino acid into
GFP created a new “Gold” fluorescent protein with a max 574 nm.
Protein Glycosylation
Sugar–Peptide Bonds
Sugar–Amino Acid Linkages of Glycoproteins
Type of bond
N-glycosyl
O-glycosyl
C-mannosylation
Phosphoglycosyl
Glypiation
Linkage
Sugar
Configuration
Asn
GlcNAc
Asn
Glc
Asn
GalNAc
Asn
Rha
Arg
Glc
Ser/Thr
GalNAc
Ser/Thr
GlcNAc
Ser/Thr
Gal
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
Pse
Ser
Glc
Ser
FucNAc
Ser
Xyl
Ser
Gal
Thr
Man
Thr
GlcNAc
Thr
Glc
Thr
Gal
Hyli
Gal
Hyp
Ara
Hyp
Gal
Hyp
GlcNAc
Tyr
Glc
Tyr
Glc
Tyr
Gal
Trp
Man
Ser
GlcNAc
Ser
Man
Ser
Fuc
Ser
Xyl
Pr-C-(O)-EthN-6-P-Man
β
β
*
*
β
α
β
α
α
α
α
β
β
β
α
α
α
*
*
β
β
β
*
α
β
β
α
α-1-P
α-1-P
β-1-P
*-1-P
Examples
Ovalbumin, fetuin, insulin receptor
Laminin, H. halobium S-layer
H. halobium S-layer
S. sanguis cell wall
Sweet corn amylogenin
Mucins, fetuin, glycophorin
Nuclear and cytoplasmic proteins
Earthworm collagen, B. cellulosoleum
Yeast mannoproteins
Coagulation and fibrinolytic factors
C. jejuni flagellins
Coagulation factors
P. aeruginosa pili
Proteoglycans
Cell walls of plants
M. tuberculosis secreted glycoproteins
Dictyosteliumh, T. cruzi
Rho proteins (GTPases)
H. halobium S-layer, vent worm collagen
Collagen, C1q complement
Potato lectin
Wheat endosperm
Dictyostelium cytoplasmic proteins
Muscle and liver glycogenin
C. thermohydrosulfuricum S-layer
T. thermohydrosulfuricus S-layer
RNase 2, interleukin 12, properdin
Dictyostelium proteinases
L. mexicana acid phosphatase
Dictyostelium proteins
T. cruzi cell surface
T. brucei VSG, Thy-1, Sulfolobus proteins
Consensus Squences or Glycosylation Motifs for the Formation of
Glycopeptide Bonds
Glycopeptide bond
GlcNAc-β-Asn
Glc-β-Asn
GalNAc-α-Ser/Thr
GlcNAc-α-Thr
GlcNAc-β-Ser/Thr
Man-α-Ser/Thr
Fuc-α-Ser/Thr
Glc-β-Ser
Xyl-β-Ser
Glc/GlcNAc-Thr
Gal-Thr
Gal-β-Hyl
Ara-α-Hyp
GlcNAc-Hyp
Glc-α-Tyr
GlcNAc-α-1-P-Ser
Man-α-1-P-Ser
Man-α-Trpf
Man-6-P-EthN-C(O)-Pr
Consensus sequence or peptide domain
Asn-X-Ser/Thr (X = any amino acid except Pro)
Asn-X-Ser/Thr
Repeat domains rich in Ser, Thr, Pro, Gly, Ala in no special sequence
Thr rich domain near Pro residues
Ser/Thr rich domains near Pro, Val, Ala, Gly
Ser/Thr rich domains
EGF modules (Cys-X-X-Gly-Gly-Thr/Ser-Cys)
EGF modules (Cys-X-Ser-X-Pro-Cys)
Ser-Gly (Ala) (in the vicinity of one or more acidic residues)
Rho: Thr-37d; Ras, Rac and Cdc42: Thr-35
Gly-X-Thr (X = Ala, Arg, Pro, Hyp, Ser) (vent worm)
Collagen repeats (X-Hyl-Gly)
Repetitive Hyp rich domains (e.g., Lys-Pro-Hyp-Hyp-Val)
Skp1: Hyp-143d
Glycogenin: Tyr-194d
Ser rich domains (e.g., Ala-Ser-Ser-Ala)
Ser rich repeat domains
Trp-X-X-Trp
GPI attached after cleavage of C-terminal peptide
The Precursor of N-Glycosylation:
In vivo Synthesis of Glc3Man9GlcNAc2-P-P-dolichol
α; b; c, etc indicate the order of addition of the monosaccharide units for in
vivo synthesis of Glc3Man9GlcNAc2-P-P-dolichol.
N-Glycosylation
1. The GlcNAc-β-Asn bond is established through the cotranslational transfer of the
dolichol-linked oligosaccharide which subsequently undergoes processing to the
large array of N-linked carbohydrate units.
2. A consensus sequence, Asn-X-Ser/Thr, was postulated and supported by numerous
studies employing structural, mutagenic, and in vitro approaches.
3. Although the Asn-X-Ser/Thr sequence occurs frequently in proteins, it does not
necessarily indicate the actual presence of an N-glycosidic linkage, most probably
due to conformational factors.
4. Replacement of Thr by Ser residues resulted in a pronounced decrease in glycosyl
transfer. The Ser or Thr is required for a hydrogen-bond donor function in enzyme
binding, although cysteine could take the place of the hydroxyamino acid.
5. The negative effect of Pro as the X amino acid has been attributed to its interference
with the ability of the peptide chain to adopt a loop conformation.
6. The oligosaccharyltransferase has been isolated and shown to be a heterooligomeric
ER membrane complex.
O-Glycosylation I:
The GalNAc-α-Ser/Thr Bond
1. The enzymes for biosynthesis of the GalNAc-α-Ser/Thr bond are a family of at least
nine GalNAc-transferases.
2. These enzymes work in concert in a hierarchical manner to form the clustered
Ser/Thr-linked oligosaccharides that occur in the “mucin”-type of glycoprotein.
3. Several of these enzymes have been cloned and they are distinct gene products and
distributed on different chromosomes.
4. Although these enzymes act on characteristic peptide regions, no specific consensus
sequence has been identified. Because they are assayed without prior separation,
overlapping distinct substrate specificities may be masked.
5. This linkage is found in clusters of Ser/Thr residues with a β-turn near Pro and at a
distance from charged amino acids.
6. In vitro studies suggest that Thr is favored over Ser for α-GalNAc modification.
7. The α-GalNAc-transfer occurs in the cis-Golgi.
O-Glycosylation II:
The GlcNAc-β-Ser/Thr bond
1. Attachment of GlcNAc-β-Ser/Thr to eukaryotic nuclear and cytosolic proteins is as
dynamic and possibly as abundant as Ser/Thr phosphorylation.
2. Known GlcNAc-β-Ser/Thr attached proteins include cytoskeletal proteins and their
regulatory proteins; viral proteins; nuclear-pore, heat-shock, tumor-suppressor, and
nuclearoncogene proteins; RNA polymerase II catalytic subunit; and a multitude of
transcription factors. Although functionally diverse, all of these proteins are also
phosphoproteins.
3. Most GlcNAc-β-Ser/Thr attached proteins form highly regulated multimeric
associations.
4. GlcNAc-transferase is localized outside of the channels of the secretory apparatus and
has been purified and cloned.
5. The Ser/Thr residues which GlcNAc-transferase glycosylates are identical to those that
can undergo O-phosphorylation, suggesting that there is a reciprocal relationship
between these two modifications in a potential regulatory cycle in which cytosolic βN-acetylglucosaminidase also plays a key role.
6. Although no specific amino acid consensus sequence has as yet been found, some
information relating to the polypeptide domains that it favors has been obtained.
The Role of Protein Glucosylation in Protein Folding
If not properly folded, the
glycoprotein is liberated by GII
from the Cnx/Crt anchor and
reglucosylated by GT to allow
rebinding of the glycoprotein to
the lectins.
Unfolded glycoproteins with
monoglucosylated oligosaccharides
is recognized by two lectins,
membrane-bound calnexin (Cnx) and
its soluble homolog, calreticulin
(Crt).
Upon adoption of the native
structure, the glycoprotein is
released from Cnx/Crt by
GII and not reglucosylated
by GT.
Protein-linked Glc3Man9GlcNAc2
is partially deglucosylated to the
monoglucosylated derivative by GI
and GII.
The Importance of Protein Glucosylation in
Protein Folding
1. The lectin-monoglucosylated oligosaccharide interaction is one of the
alternative ways by which cells retain improperly folded glycoproteins in
ER.
2. Although it decreases the folding rate, it 1) increases folding efficiency; 2)
prevents premature glycoprotein oligomerization and degradation; 3)
suppresses formation of nonnative disulfide bonds
3. This allows interaction of protein moieties of folding glycoproteins with
chaperones that assists in further folding.
Phosphoglycosylation
1. The attachment of a sugar to the polypeptide chain through a phosphodiester bridge
has been termed phosphoglycosylation,
2. GlcNAc-1-phosphotransferase was partially purified and localized in the Golgi
compartment.
3. GlcNAc-1-phosphotransferase recognizes Ser-containing peptides of various
proteins among which cysteine proteinases are the most prominent.
4. No single specific motif was observed. However, the transfers occur in Ser-rich
domains in which the flanking Ala residues preferentially influence
phosphoglycosylation.
5. GlcNAc-1-phosphotransferase does not phosphoglycosylate Thr residues.
6. Man-1-phosphotransferase has been characterized and adds Man-α-1-phosphate to
Ser residues in domains rich in Ser. It does not act on Thr and its action is promoted
by flanking Asp and Glu residues.
C-Mannosylation
1. The enzyme which links C-1 of mannose to the C-2 atom of the indole ring
of Trp has been studied in rat liver microsomes.
2. The glycosyl donor in this reaction is Dol-P-Man.
3. The dependence of the C-mannosylation on Dol-P-Man strongly suggests
that it takes place in the ER, where all known Dol-P-Man-dependent
reactions are localized.
4. The recognition signal for C-mannosylation has been assigned to a Trp-X-XTrp sequence in which the first Trp becomes glycosylated.
5. The Trp at position +3 is important for the glycosylation as the transfer
activity was abolished when this amino acid was mutated to Ala and reduced
to one-third when replaced by Phe.
6. A survey of protein databases has indicated that the Trp-X-X-Trp consensus
sequence is present in 336 mammalian proteins, suggesting the possibility
that C-mannosylation may occur quite frequently in higher eukaryotes.
Protein Phosphorylation
Historyic Events of Protein
Phosphorylation
1. In the 1950s, it was discovered that the metabolic enzyme
phosphorylase, responsible for the conversion of glycogen to
glucose- 1-P, existed in an inactive (phosphorylase b) and an
active (phosphorylase a) forms.
2. This conversion is made by phosphorylase kinase which
catalyzes phosphorylation of phosphorylase and render it
fully active.
3. Phosphorylase kinase was itself activated by protein kinase A
(PKA) and the concept of protein phosphorylation as a key
regulatory mark was born.
Importance of Protein Phosphorylation
1. Most cellular processes and cell signaling pathways are
regulated by protein phosphorylation catalyzed by protein
kinases.
2. Protein kinases are regulated by inhibitory or activating
protein partners, phosphorylation, cellular localization
limiting availability of substrates and activators, protein
degradation, and gene transcription.
3. Phosphorylation can result in enzyme activation, enzyme
inhibition, the creation of recognition sites for recruitment of
other proteins, and transitions in protein state from order to
disorder or disorder to order.
The Enzymes for Protein Phosphorylation:
Protein Kinases
1. More than 518 human protein kinases were recognized through their
conserved sequence motifs of which 478 protein kinases are typical and 40
are atypical.
2. The typical kinases are divided into those that phosphorylate serine or
threonine residues (388 kinases) and those that phosphorylate tyrosine
residues (90 kinases).
3. Atypical kinases have biochemical kinase activity but lack sequence
similarity to the conventional kinases.
4. Unique kinase domain structures (170) from humans or closely related
orthologs had been determined.
5. A characteristic feature of the protein kinase family is the different
structures that they adopt between the active and inactive states.
6. Adoption of the active state occurs in response to specific signaling events,
which are transduced via kinase associated regulatory domains and by
phosphorylation of the kinase domain.
Catalytic Mechanism of Protein Kinases
Protein kinases catalyze the transfer of a phosphoryl group from
the γ-phosphate of ATP to the hydroxyl group of serine, threonine,
or tyrosine residues of proteins by the reaction scheme:
Protein-OH + ATP4−.Mg2+ →
Protein-O-PO3 2−+ ADP3− .Mg2+ + H+
Most protein kinases show specificity for the local region around
the site of phosphorylation where certain residues are required for
recognition.
Protein Kinases and Their Preferred Substrate
Specificities
Substrate recognition at the catalytic site involves specific residues in the region near
the site of phosphorylation. Association between kinase and substrate is often low
affinity, and greater stability is achieved through docking sites that are remote from the
catalytic site.
Protein Kinase Catalysis
1. The kinase reaction proceeds with an inline mechanism in which the
attacking group (serine, threonine, or tyrosine OH) comes in opposite to
the leaving group (phosphate ester oxygen), leading to inversion of
configuration at the phosphorus.
2. The catalytic step was fast (k3 ∼ 300–500 s−1), and the release of
products relatively slow (k4 ∼ 20–30 s−1).
3. Thus, the rate-limiting step is the release of products, i.e., ADP and
phosphorylated proteins.
Protein Kinase Modulation
1. Phospho-signaling is a rapid action. Changes in specific
phosphorylation targets can be detected within minutes after
exposure stimuli.
2. When epidermal growth factor (EGF) is added to the media,
greater than 500 proteins undergo phosphorylation changes
by 5 min.
3. The specific kinetic details of these cellular phosphoryl
transfer reactions are critical to their macroscopic effects on
gene regulation, cell shape, and cell growth, which occur
over longer timescales.
Protein Methylation
Methylase-Catalyzed Reactions
Chromatin and Histone Modifications
The eukaryotic DNA is compacted within the cell
nucleus through its interactions with histone proteins,
forming the nucleosomes.
The histone N-terminal tails protrude
outward beyond the gyres of DNA.
Many of the amino acid residues within the
histone tails can be posttranslationally
modified, providing a landing pad for a
diverse array of transcription factors,
chromatin remodelers, and DNA-interacting
proteins to regulate gene expression and other
DNA-dependent processes.
An Example of Protein Methylation:
Histone H3 Lysine 4 (H3K4) Methylation
1. Methylation of histone Lys plays a crucial role in the regulation of key
biological processes, such as cell cycle progression, transcription, and DNA
repair .
2. In yeast, histone H3K4 methylation is carried out by SET [Su (var),
Enhancer of Zeste, and Trithorax] domain-containing enzymes. The Set1
family protein forms a multiprotein complex named COMPASS (COMplex
of Proteins ASsociated with Set1) in yeast.
3. Set1/COMPASS was the first identified histone H3K4 methylase capable of
mono-, di-, and trimethylating H3K4.
4. In addition to the evolutionarily conserved SET domain located at the C
terminus of Set1, most associating subunits are also conserved from yeast
to human.
5. The histone H2B monoubiquitinase Rad6/Bre1 is required for proper H3K4
trimethylations.
Protein Acetylation
N-Acetylation Reactions
N-Terminal Acetylation in Prokaryotes and Eukaryotes
Acetylation Sites in Histones
Hyperacetylated Chromatin Domains
1. In eukaryotes, the genome is packaged into two general types of
chromatin: heterochromatin, which appears compact or
condensed throughout the cell cycle, and euchromatin, which
appears condensed only prior to mitosis.
2. A small number of loci that exhibit covalent histone
modifications by histone acetyltransferases (HAT), such as
hyperacetylation.
3. The hyperacetylated domains occur exclusively at loci containing
highly expressed, tissue-specific genes, and that they are involved
in the activation of these genes.
Hperacetylated Domain of Heterochromatin
A.A complex is nucleated at a
regulatory sequence (blue line).
B. This complex includes a HAT which
modifies nearby nucleosomes.
C. Modified nucleosomes in turn
represent high affinity binding sites
for a subset of the complex, resulting
in the progressive spread of the
complex.
D.Additional sequences may be bound
by factors that block the further
spread of the complex and thus serve
as boundaries (yellow oval).
Protein Acetylation in Prokaryotes
1. Protein acetylation plays a critical regulatory role in eukaryotes but
prokaryotes also have the capacity to acetylate both the N-terminal
residues and the side chain of Lys and is widespread for regulation of
fundamental cellular processes.
2. Lys acetylation in particular can occur in proteins involved in transcription,
translation, pathways associated with central metabolism and stress
responses.
3. Specific acetylated Lys residues map to critical regions in the 3-D of key
proteins at active sites or surfaces that dock with other major cellular
components.
4. Like phosphorylation, acetylation appears to be an ancient reversible
modification that can be present at multiple sites in proteins, thereby
potentially producing epigenetic combinatorial complexity.
5. Acetylation is particularly important in regulating central metabolism in
prokaryotes due to the requirement for acetyl-CoA and NAD+ for HAT and
HDAC, respectively.
Signals and Combinatorial Functions of Histone
Modifications
1. Alterations of chromatin structure are crucial for response to cell signaling
and for programmed gene expression in development.
2. Posttranslational histone modifications influence changes in chromatin
structure both directly and by targeting, or activating chromatin-remodeling
complexes.
3. Histone modifications intersect with cell signaling pathways to control
gene expression and can act combinatorially to enforce or reverse
epigenetic marks in chromatin.
4. Through their recognition by protein complexes with enzymatic activities,
cross talk is established between different modifications and with other
epigenetic pathways, including noncoding RNAs (ncRNAs) and DNA
methylation.