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1. Sinhhọcphântử
Bài8
TS. Lê Quang Dũng
BộmônSinhhọcTếbào
Email: [email protected]
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2. 5. MECHANISMS THAT REINFORCE CELL
MEMORY IN PLANTS AND ANIMALS
6. POST-TRANSCRIPTIONAL CONTROLS
7. REGULATION OF GENE EXPRESSION BY
NONCODING RNAs
2

3. 5. MECHANISMS THAT
REINFORCE CELL
MEMORY IN PLANTS
AND ANIMALS
3

4. Patterns of DNA Methylation Can Be
Inherited When Vertebrate Cells Divide
•The methylation of cytosine
provides a mechanism through
which gene expression patterns
can be passed on to progeny
cells.
•The DNA methylation of
cytosinehas no effect on base-
pairing.
4

5. •DNA methylationof cytosine(C) mainly occurs at CG sequences
on both strands of the DNA helix.
•Enzyme maintenance methyl transferase recognized methylated
CGsequences and methylase the CG on the complementary strand.
•As a result, the pattern of DNA methylation on the parental DNA
can be inheriteddirectly following DNA replication by the daughter
DNA strands.
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6. •DNA methylation represses transcription to
very high degrees.
•The methyl groups on methylated cytosines
lie in the major groove, preventingthe binding
of proteins.
•Histone reader and writer proteins, under
the direction of transcription regulators,
establish heterochromatina repressive form of
chromatin.
•A DNA methylase is attracted by the histone
reader and methylates nearby cytosines in DNA,
which are, in turn, bound by DNA methyl-
binding proteins.
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7. CG-Rich Islands Are Associated
with Many Genes in Mammals
•Deamination of an unmethylated
Cgives rise to U, which is not
normally present in DNA and thus is
correctedby the DNA repair enzyme
uracil DNA glycosylase, back to a C.
•However, deamination of a
methylated Cturn it into a T
nucleotidein the DNA.
•-Although a special repair system
exists to remove these mutant T
nucleotides, many of these mutant
T escapedetection, eliminating
methylated C out over evolutionary
time.
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8. •During the course of evolution,
more than three out of every four CGs
have been lostin this way.
•In CG island, the concentrationof
CG dinucleotideis 10x higher than the
average.
•CG islands are located in promoters
of housekeepinggenes, also remain
unmethylatedstate.
•Mutation at CG islands is prevented
by sequence-specificDNA-binding
proteinswhich protectDNA from
methyl transferase.
•These proteinsalso recruitDNA
demethylases, which convert5-methyl
C to hydroxy-methyl C which is
replaced by C later.
8

9. Genomic Imprinting Is
Based on DNA
Methylation
•-Mammalian cells are diploid,
containing one set of genes
inherited from the father and one
set from the mother.
•Genomic imprinting is the
expression of a small minority of
genes inherited from either from
motheror father.
9

10. •-Intheearlyembryo,genesare
markedbymethylationwhether
theywerea spermoranegg
chromosome.
•-Duringgerm-cellformation,but
beforemeiosis,theimprintsare
erasedandthen,muchlaterin
germ-celldevelopment,theyare
reimposedinasex-specificpattern.
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11. •In the case of Igf2, methylation
of an insulator element on the
paternalchromosomeblocks its
function and allows distant cis-
regulatory sequences to activate
transcription of the Igf2 gene.
•On the maternal chromosome,
the insulatoris not methylatedand
the Igf2 gene is therefore not
transcribed.
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12. •MaternalKcnq1geneis
methylated,blockingthesynthesis
ofthelncRNA butallows
transcriptionoftheprotein-coding
gene.
•Inunmethylatedpaternalallele,
lncRNAismadeandattachedto
RNApolymerase.ThislncRNAthen
recruitshistone-modifyingand
DNA-methylatingenzymes to
represschromatin,whichsilences
theprotein-codinggene.
12

13. Chromosome-Wide
Alterations in Chromatin
Structure Can Be Inherited
•-Femaleshave twoXchromosomes,
whereas maleshave one Xand one Y
chromosome.
•-The X chromosome is large, containing
more than a thousand genes, whereas the Y
chromosome is smallwith less than 100 genes.
•Mammals have evolved a dosage
compensation mechanism to equalizethe
dosage of X-chromosome gene between males
and females.
•-Two X chromosomes can coexistwithin the
same nucleus, exposed to the same
transcription regulators, yet differ entirely in
their expression.

14. •When female embryo starts
developing, one of the two X
chromosomesin each cell becomes
highly condensed into a type of
heterochromatin.
•Once either Xpor Xmhas been
inactivated, it remains silent
throughout all subsequent cell
divisions of that cell and its
progeny.

15. •Using genetic engineeringtechniques, the
germ line of a mouse was modified so that one
copy of the X chromosome (if active) makes a
green fluorescent protein and the other a red
fluorescent protein.
•These clonal groups are distributedin small
clustersin the adult animal because sister cells
tend to remain close togetherduring later
stages of development

16. •X-chromosome inactivation is initiated
and spreads from X-inactivation center
(XIC) near the middle of the X
chromosome.
•A lncRNA (called Xist)is a transcribed
from XIC , which is expressed solely from
the inactive X chromosome.
•XistRNA spreads by a “hand-over-
hand” mechanism from the XIC over the
entire chromosome and directs gene
silencing.
•It likely involves recruitment of
histone-modifying enzymes and other
proteins to form a repressive complexes.

17. Epigenetic Mechanisms
Ensure That Stable Patterns of
Gene Expression Can Be
Transmitted to Daughter Cells
•The ability of a daughter cell to
retain a memory of the gene
expression patterns that were
present in the parent cell is an
example of epigenetic inheritance.
•A maintenance methylasecan
propagate specific patterns of
cytosine methylation.
•Histone modifying enzyme that
replicates the same modification
to maintaineuchromatin or
heterochromatin structure of
daughter cells.

18. •Positive feedback loops are
probably the most common
form of cell memory.
•Proteins can form self-
propagating prions. If these
proteins are involved in gene
expression, they can transmit
patterns of gene expression to
daughter cells.

19. 6. POST-
TRANSCRIPTIONAL
CONTROLS

20. Transcription Attenuation
Causes the Premature
Termination of Some RNA
Molecules
•The expression of some genes is
inhibitedby premature termination
of transcription, a phenomenon
called transcription attenuation.
•RNA chain adopts a structure
that causes it to interact with the
RNA polymerase in such a way as to
abortits transcription.

21. Riboswitches Probably
Represent Ancient
Forms of Gene Control
•Riboswitches, common in
bacteria, are short sequences of
RNA that change their
conformation to regulate gene
expression.
•Each riboswitch recognizesa
specific small molecule and the
resulting conformational change is
used to regulate gene expression.

22. •When guaninelevels in cells are low, an elongating
RNA polymerase transcribesthe purine biosynthetic
geneswhich is the gene needed for guanine synthesis.
•When guanine is abundant, it binds the riboswitch,
causing it to undergo a conformational change that
forces the RNA polymerase to terminate transcription.

23. Alternative RNA Splicing Can
Produce Different Forms of a
Protein from the Same Gene
•RNA splicing shortens the
transcripts of many eukaryotic genes
by removing the intron sequences
from the mRNA precursor.
•-Additionally, alternative RNA
splicing is the process in which cell
can splice an RNA transcript
differentlyand thereby make
different polypeptide chains from
the same gene.

24. Drosophilagene
may produce as
many as 38,000
different proteins
from a single
gene through
alternative
splicing.

25. •RNA splicing can be regulated
negatively, as a regulatory
molecule preventsthe splicing
machineryfrom gaining access
to a particular splice site on the
RNA.
•Splicingcan also be regulated
positively, as a regulatory
molecule directs the splicing
machinery to its splice site.

26. A Change in the Site of RNA Transcript Cleavage
and Poly-A Addition Can Change the C-terminus
of a Protein
•Different sites of RNA cleavage
can create a variety of proteins from
one single gene.
•One example is development of
B lymphocytes.
•At low level of CstFprotein,
unstimulated B lymphocyte
produces long version of target
protein which serves as membrane-
bound receptor.
•At high level of CstFprotein, RNA
cleavage occurs at the weak site of
the target gene, producing short
transcript of secreted antibodies.

27. RNA Editing Can Change the
Meaning of the RNA Message
•In animals, two principal types of mRNA
editing occur: the deaminationof adenine
to inosine (A-to-I editing), and cytosine to
uracil (C-to-U editing).
•The process of A-to-I editing is catalyzed
by enzymes called ADARs(adenosine
deaminases acting on RNA).
•ADARs recognize a double-stranded
RNA formed through base-pairing and edit
specific A base.
•An important example of A-to-I editing
is transmitter-gated ion channel in the
brain, which can alter the Ca
2
+ permeability
of the chanel. Mutant mice without this
edit are prone to have epileptic seizures.

28. •C-to-Uediting is very important in
apolipoprotein B gene creating two
versions of protein in liver cells and
gut cells.
•In liver cells, editing enzyme is not
expressed, therefore the full-length
apolipoprotein Bis produced to serve
in liver cells.
•In gut cells, the mRNA for
apolipoprotein B undergoes a C-to-U
edit to form a prematurestop codon,
producing a shorter form of the
protein.

29. RNA Transport from
the Nucleus Can Be
Regulated
•mRNA issynthesized,
processedtoformmatureRNA
inthenucleus,beforebeing
transportedintocytoplasm.
•Onlyone-twentiethofRNA
synthesizedeverleavesthe
nucleus.
•TherestoftheRNAincluding
excisedintronsaredegradedin
thenucleus.

30. •Inside the cell, HIV virus directs the formation of a double-
stranded DNA copy of its genome, then insert it into the genome of
the host.
•Then the viral DNA can be transcribed to form one long RNA
molecule by the host cell’s RNA polymerase II.
•This long transcript is then splicedin many different ways to
produce over 30 different species of mRNA, which in turn are
translated into a variety of different proteins.

31. •HIV virus createsprogeny virus by sending and packaging
unsplicedviral transcripts into viral capsids in the cytosol.
However, the host cell blocksthe nuclear exportof unspliced
RNAstherefore presents a special problem for HIV.
•The virus overcomesthis obstacle by encoding Rev protein
that bindsto RNA sequence called Rev responsive element
(RRE).
•In the early phase, fully spliced RNAs containing coding
sequences for Rev are exportedfrom the nucleus and translated
into Rev protein.

32. •Inthelatephase,sufficientRevproteinhasaccumulatedand
beentransportedintothenucleus,thenbindtotheRRE
sequencesonunsplicedviralRNAs.
•Afterward,Revproteininteractswithanuclearexport
receptor(Crm1),directingtheunsplicedviralRNAsthrough
nuclearporesintothecytosoldespitethepresenceofintron
sequences.
•Inthecytosol,manyoftheseRNAsaretranslatedinto
protein,andthefull-lengthtranscriptsarepackagedintonew
viralparticles.

33. Some mRNAs Are Localized to
Specific Regions of the Cytosol
•Priortotranslation,manymRNAs
arethemselvesdirectedtospecific
sitesincytosolwheretheencoded
proteinisneeded.
•SeveralmechanismsformRNA
localizationareemployed,allof
whichrequirespecificsignalsinthe
mRNAitselfinthe3′untranslated
region(UTR)betweenthestop
codonandthestartofthepoly-A
tail.

34. The 5′ and 3′ Untranslated Regions of mRNAs Control
Their Translation
•In bacterial mRNAs, the Shine–Dalgarno sequence, is always found upstreamof the
initiating AUG codon in the 5’ end to control translation.
•In eukaryotes, translational repressorscan bind to the 5′ end and 3’ end of the mRNA
and thereby inhibit translation initiation.

35. The Phosphorylation of an Initiation Factor Regulates
Protein Synthesis Globally
Eukaryotic translation depends on factor eIF2. The factor binds to GTP and small subunit ribosome
to screen for target AUG codon. Once AUG found, eIF2 hydrolyzesGTP to GDP, and separatefrom
the small subunit to allow the large subunit joins and start translation.
As eIF2-GDP is very stable, another factor eIF2B, is required to release GDPso that eIF2 can be
reused. However, eIF2B cannot separateGDP from phosphorylated eIF2, therefore
phosphorylation of eIF2 can significantly slow down protein synthesis.

36. Internal Ribosome
Entry Sites Provide
Opportunities for
Translational Control
•Cellscaninitiatetranslationatpositionsdistantfromthe5′
endofthemRNA,usingaspecializedtypeofRNAsequence
calledaninternalribosomeentrysite(IRES)
•IRESsarehundredsofnucleotidesinlengthandadapt
specificstructuresthatbindnotallbutsomeoftheproteins
requiredfortranslation.Thisallowsthemtobypasstheneedfor
a5′capstructureandthetranslationinitiationfactorthat
recognizesit,eIF4E

37. Changes in mRNA Stability
Can Regulate Gene
Expression
•mRNAsinabacterialcellarevery
unstable,havinghalf-livesoflessthana
coupleofminutestoallowsbacteriato
adaptquicklytoenvironmentalchanges.
themRNAsineukaryoticcellsaremore
stablewithlongerhalf-lives.
•AsthemRNAreachesthecytosol,its
poly-Atailisgraduallyshortenedbyan
exonucleaseandkeepdegradingfurther
intothecodingsequences.Oncethe
poly-Atailisreducedtoacriticallength
(25nu),the5′capisremovedandexpose
mRNAforrapiddegradation.

38. •The specific sequences of each
mRNA determine how fast
degradationwill happen.
•The 3′ UTRoften carry binding
sites for specific proteins that
increase or decrease the rate of
poly-A shortening, decapping, or 3′-
to-5′ degrada?on.

39. •Specific endonucleasesidentifyspecific
nucleo?de sequences in the 3′ UTRs and cleave
the mRNA internally, effectively decappingone
end and removing the poly-A tail so that both
halves are rapidly degraded.
•For example, iron-sensitiveRNA-binding
protein aconitaseto controlthe stabilityof iron-
transporting protein transferrin.
•Aconitasebindsto the 3′ UTR of the
transferrin mRNA to blockendonucleolytic
cleavage and protect transferrin mRNA.
•With iron, aconitaseis releasedfrom the
mRNA, exposing the cleavage site and thereby
decreasingthe stability of the transferrin mRNA.

40. Regulation of mRNA
Stability Involves P-bodies
and Stress Granules
•In the cytosol, large aggregates of
proteins and nucleic acids that work
together are often held in P-bodies
which is an “organelles” not
surrounded by membranes.
•Within P-bodies,decappingand
RNA degradation take place to
clean up redundant materials in the
cytosol.

41. •An mRNAmolecule released
from the nucleus can be actively
translated (center), stored in stress
granules (right), or degraded in P-
bodies (left).
•Clearly, once a cell has made the
large investment in producing a
properly processed mRNA molecule,
it carefully controls its subsequent
fate.

42. 7. REGULATION OF
GENE EXPRESSION BY
NONCODING RNAs

43. Small Noncoding
RNA Transcripts
Regulate Many
Animal and Plant
Genes Through
RNA Interference
•Short single-stranded RNAs (20–30nucleotides) serve as
guideRNAs that selectively reorganize and bind other RNAs in
the cell.
•Three classes of small noncoding RNAs work in this way:
microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-
interacting RNAs (piRNAs)
•-Once the target mRNA identified, the small noncoding RNAs
can: inhibititstranslation,catalyzedestruction of target RNA, or
induce formation of heterochromatin to repress transcription.

44. miRNAs
Regulate
mRNA
Translation
and Stability
•Over 1000 miRNAs are in human genome, regulatingabout
one-thirdof protein-coding genes.
•miRNAprecursors are synthesized by RNA polymerase II and
are capped and polyadenylated. They then undergo further
processing, after which the miRNA (23 nucleotides long) is
assembled with a set of proteins to form an RNA-induced
silencing complex or RISC.

45. •-The function of RISC complex
greatly dependson Argonaute
protein, a component of RISC,
binding to to 5’ end of miRNA to
base-pair to its target RNA.

46. miRNA complex goes through:
•Extensivematch: effectively
degrade mRNA
•Less extensive match: repress
translationand move RNAs to P-
bodieswhere they are eventually
degraded

47. RNA Interference Can
Direct Heterochromatin
Formation
•ThesmallinterferingRNAs
(siRNAs)producedbytheDicer
proteinareassembledwithagroup
ofproteinstoformtheRITS(RNA-
inducedtranscriptionalsilencing)
complex.
•TheRITScomplexattracts
proteinsthatcovalentlymodify
nearbyhistones,leadingtothe
formationofheterochromatinto
preventfurthertranscription.

48. Bacteria Use Small Noncoding RNAs to Protect
Themselves from Viruses
-Similarto miRNAsystem, CRISPR system is utilized to seek out and destroydouble-
stranded DNA molecules, rather than single-stranded RNA molecules.
-In the first, viral DNA sequences are integratedinto special regionsof the bacterial
genome known as CRISPR.
-In the second step, the CRISPR locus is transcribedto produce a long RNA molecule,
which is then processed into the much shorter(approximately 30 nucleotides) crRNAs.
-In the third step, crRNAscomplexedwith Cas(CRISPR-associated) proteins seek out
complementary viral DNA sequences and direct their destructionby nucleases.

49. Long Noncoding RNAs Have Diverse Functions in the
Cell