rucime of eukaryotic morne, its replication nů gene expression
Biology
Doc. RNDr. Jan Hosek, Ph.D. hosek@mail.muni.cz
Department of Molecular Pharmacy FaF Ml)
https://www.youtube.com/watch?v=7Hk9jct2ozY&ab
channel=WEHImovies
Structure ©f eukatyotíc
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Genom of eukaryotic organisms
Animal cells: nucleus and mitochondria
Plant cells: nucleus, mitochondria and chloroplasts
> chromosomal (AKA nuclear) DNA (nDNA)
> mitochondrial DNA (mtDNA)
> chloroplast DNA (ctDNA)
> plasmids
Chromatin
> Stainable material, which forms the nucleus of eukaryotic cells
> dsDNA, histories, nonhistones
According to the ability to be stained by basic dyes and degree of condensation we distinguish:
> euchromatin - weakly stainable, decondensed, "transcriptionally active"
> heterochromatin - strongly stainable, condensed, "transcriptionally inactive"
heterochromatin
https://www.studyblue.com/notes/note/ri/3-chromosomes/deck/4743713
Heterochromatin
Constitutive
- Constantly in heterochromatin stage
- centromeres and telomeres
- One of X chromosome in women
Facultative
- Switches between heterochromatin and euchromatin on the base of oncogenetic development of organism
Chromatin condensation
Metaphase eh romosome
1400 nm
V
1) Basic structure = interphase = decondensed 10-nm chromatin fiber (beads-on-a-string)
2) 30-nm chromatin fibre
3) Chromatin in mitotic phase = mitotic chromosomes
Condensed scaffold-associated form
Extended scaffold-SSSOCiflted form
30-nm chromatin fiber of packed nucleosomes
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- Chromosome scaffold
300 nm
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"Beads-on-a-string" form of chromatin
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Short region of DNA double-helix
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2 nm
Chromatin condensation
fl^MMMM, „naked" dsDNA
"beads-on-a-string" form nucleosomes
30 nm solenoid
relaxed form of chromosome
condensed region of chromosome
mitotic chromosome
Chromatin components
1) Histones
> Centre is globular, ends are flexible and filamentous
> High content of arginine and histidine
> 5 species = H1, H2A, H2B, H3 and H4
2) Nonhistones
> RNA polymerase and other enzymes usable in transcription
> HMG1 and HMG2 - bind to unusual DNA structures
> HMG3 a HMG4 - bind to histone core especially in transcriptionally active regions
Nukleosome
> The basic unit of chromatin
> octamer of histones (H2A, H2B, H3, H4)2
> One molecule of histone H1
> DNA segment 200 bp long, which is wound about 2 times around the octamer of histones
Nucleosome structure
H2A- yellow, H2B red, H3 blue, H4 green
Nature 389: 251-260 (1997)
Nucleosome fibre
> 10nm chromatin fibre
> its individual items form nucleosome cores connected by long linear dsDNA
> visible by microscope
-H1
+ H1
H1
30nm chromatin fibre
30nm chromatin fibre
> It is created by the condensation of nucleosome fibre caused by histone H1
> It binds to protein scaffold (nonhistones, e.g. topoisomerase II)
Chromatin domains
> loops of 30nm chromatin fibre attached to protein scaffold
> there is one molecule of topoisomerase II in base of each loop = change of topology during replication and transcription
> each domain has one ori locus
DNA protein scaffold connecting region
Mitotic chromosomes
> They originate by condensation of 30nm chromatin fibres
> They are formed during mitosis or meiosis
> Condensation of 30nm to 600-700nm chromatin fibres, which constitute the structure of chromosomes
> In chromosomes, the chromatin is in the stage of the highest condensation and is transcriptionally inactive
Mitotic chromosomes
sister chromatids
Organisation of chromatins in nucleu
> Localisation of chromosomes is not accidental
> Crowding of region with similar function or activity
DOI: 10.1038/nsmb.2474
> replication of mitochondrial and chloroplat DNA
> replication of nuclear chromosomes
> semiconservative and semidiscontinuous
> initiation, elongation, and termination
> only in S phase of the cellular cycle
transcription,
translation,
metabolism
In contrast to prokaryotic cells the eukaryotic replication proceeds on several places in time
Chromosome is a couple of replicons, it has more on sequences (mammals 30.000-50.000)
Euchromatin replicates earlier to heterochromatin
0
old
nukleosomes
•oc<.*o
oo0o°-
strand
newly synthesises nucleosomes
beginning of bidirectional replication
Eukaryotic DNA-polymerase: a, p,   5 and £
lagging strand
PCNA Pol 5
primase
Leading strand template
RPA
Helicase (T antigen)
RNA primer Pol a
DNA Ligase
T-TT
Topoisomerase
A
FEN-I Lagging strand template
RNase HI
http ://yxsj .baiduyy.com/
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SINS
Mcm2-7 helicase
leading strand
PCNA
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G1-phase
Mcm2-7
S-phase
Cyclin Dependent Kinase Cdc7 kinase (» assembty factors, ♦Pol fps or. 0)
http://www.ppu.mrc.ac.uk/researc h/?pid= 1012&sub 1 =research
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template DNA
leading strand
3 5
+ 3
RNA primer removing
Okazaki fragments
replication of spacers
incompletely replicated DNA, missing nucleotides y
the end replication problem
Telomerase = ribonukleoprotein - RNA acts as a template, protein has catalytic function
5*
5'-
anno
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Z7
Telomerase elongates the 3-end
Formation of hairpin and RNA primer
Replication of complementary strand and removing of hairpin
ttgggg 3- Telomere Template
ttggggttgggg
1. Hairpin created; RNA Primer added
Missing DNA on . _  lagging strand
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5' 3-
RNA GGGG
Primer 2.Hairpin extended;
RNA primer removed
. Telomerase with its own RNA template
ttggggttgggg
■5'
AAGGGG
ttggggttgggg
aaccccaagggg
ttgggg
3. Gaptilled
4. Hairpin removed
_ Replicated Telomere
AACCCC
https://www.ndsu.edu/pubweb/~mcclean/plsc431/eukar ychrom/eukaryo3.htm
1. End is unreplicated.
2. Telomerase extends unreplicated end.
3. Again, telomerase extends unreplicated end.
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4. Lagging strand is completed.
Sliding i
O2011 Pwson Educatwn. inc
http://masteringyourwaytomedschool.blogspot.ez/p/bio-1000-dna-shortening.html
TABLE 11.5
Telomeric Repeat Sequences Within Selected Organisms
Group Examples TGlomGric Repeat Sequence
Mammals	Humans	TTAGGG
Slime molds	Physarum, Didymium	TTAGGG
	Dtctyosteiium	
Filamentous fungi	Neurospora	TTAGGG
Budding yeast	Saccharomyces cerevisiae	TG(1-3)
Ciliates	Tetrahymena	TTGGGG
	Paramecium	TTGGG(T/G)
	Euplotes	IIIIGGGG
Higher plants	Arabtdopsis	TTTAGGG
http://reasonandscience.heavenforum.org/t2263-the-telomerase-enzyme
Fig. 2 Mammalian telomeres
Telomere t-loop
Strand invasion of 3' overhang
<<
T. de Lange Science 326, 948-952 (2009)
Science
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> Primary transcripts
> heterogeneous nuclear RNA (hnRNA) = pre-mRNA forming in nucleus
> precursor ribosomal RNA (pre-rRNA)
> precursor transfer RNA (pre-tRNA)
> 5S-rRNA
> small RNA (snRNA, snoRNA, scRNA)
> Eukaryotic DNA-dependent RNA-polymerase
- RNA-polymerase , II,
> Transkription factors
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polycistronic character
trp operon in Escherichia coli-5 genes
	E	D	C	B	A	
trp mRNA
t t t   t t
B
origins of translation
5 proteins
for synthesis of Trp
3imm(§{rBßiB©[ru wďá ©ff (Btwk@^©ti
> Monocistroni character
> Contains:
> Promoter
> Leading sequence (5-UTR)
> Polyadenilation signal
> Terminator
upstream ^ ^ enhancers TATA box'
Coding region
3'UTR
1
Promoter
Exon 1
Exon 2
5' _t~l
Exon 3 /
Initial transcript
5' cap
final mRNA (in cytoplasm)
J i
Intron 2
DNA 3'
3' Poly-A tail
/
IAAAAA
T
IAAAAA
Enhancer Proximal (distal control elements)    control elements
DNA
Upstream
Poly-A signal sequence
Termination region
Exon    Intron  Exon    Intron Exon
Promoter
Primary RNA transcript
Transcription
Exon    Intron  Exon    Intron Exon
Intron RNA
RNA processing
Downstream
Cleaved 3' end 'of primary transcript
Coding segment
mRNA GHgKgHg>-^H]
Start Stop
Poly-A signal
r.VJ.V.l
3
5 Cap  5 UTR    codon    codon   3'UTR Poly-A
tail
http://nitro.biosci.arizona.edu/courses/EEB600A-2003/lectures/lecture24/lecture24.html
IV
VII
trp3
1
trp2
trp5
1
Transcription unit for synthesis of Trp in
Saccharomyces cerevisiae
= togehter 5 genes located on 4 chromosomes
> Regulatory elements necessary for transcription initiation
> Usually initiate transcription, rarely inhibit it
> Their different combination bind to the promoter, then the RNA polymerase bind to DNA strand
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> general TF
> present in all and most types of cells
> necessary to transcription initiation
> basal - low activity, minimal cell requirements
> constitutive - increase the basal activity according to cell type; basal cell requirements
> special TF
> only in cells of specific tissues and in a certain time
> applied in inducible transcription
1) Separation of transcription and translation
2) hnRNA is capped by the cap, and methylated (binding to ribosome)
3) In the 3 - region (after STOP codon) the sequence AAUAAA is present, in this location the hnRNA is digested
4) At 3 end is polyadenylated (stabilisation in cytoplasm)
5) After removing introns and joining exons it is transformed to mRNA
Binding of transcription factors on TATA box and others regulation sequences = preinitiation complex
Binding of RNAP II on preinitiation complex = closed initiation complex
Phosphorylation of CTD domain of RNAP II by trancription factor TFIIH (halicase and kinase activities) -► RNAP II activation and unwinding of dsDNA = open initiation complex
Disociation of RNAP II from TFs (except TFIIF) and start of RNA synthesis
F« -   .    _ —  CopyriQhi C The McGraw-HiH Companies. Permission required tor reproduction or display
igure 11.33
Minimal
initiation complex
Active ttt «<* transcription complex
https://www.youtube.com/watch?v=_Zyb8bpGMR0&
ab channel=ArmanHossain
Linear transcription units
Assembly of functional expression units
Expression unit Small genomes i
Coordinated expression
On
Complex genomes
Spatial transcription units
Functional organization of the nucleus
Promoter assembled from 2 promoters?
Dekker J,: Science 319,1793 -1794 (2008)
Transcription factory
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1) Terminator contains AATAAA sequence = polvadenilation signal
2) Once polyadenilation signal is transcripted into hnRNA, it is recognised by protein complex, which cut hnRNA 10-30 nt towards 3-end
3) Subsequently, RNAP II disociate from DNA and the rest of hnRNA behind the polyadenilation signal is degraded
a Termination at mRNA-coding genes in yeast
Recruitment of the CPF-CF complex
CTD «PPhosphorylated Ser2 5'
RNA cleavage and polyadenylation
Dissociation of the elongation complex
-\H-
Allosteric modeil
CPF-CF 5' complex RNA
CID-^W3 Elongation CFIAC fefljQo  factors -
TSS
£
PolJlX^
H1L
DNA Poly(A) signal
b Termination at ncRNA genes in yeast
Phosphorylated Ser5
Recruitment of the NNS complex
Dissociation of the elongation complex
-\P
RNA polyadenylation and degradation or processing
DNA
Nrdl-and Nab3-binding sites
Nature Reviews Molecular Cell Biology 16, 190-202 (2015)   Nature Reviews | Molecular Cell Biology
hnRNA modifications
> hnRNP-complexes forming
> adding cap to 5 - end
> polyadenylation of 3 - end
> splicing of hnRNA
• Proteins which specifically bind on hnRNA = hnRNP-proteins
• Proteins which specifically bind on small nuclear RNA (snRNA) = snRNP-proteins
• snRNP-proteins + snRNA = snRNP-particles
• hnRNA + hnRNP-proteins + snRNP-particles = hnRNP-complex
• snRNP-particles   bind   on   intrones   and form spliceosom, which drive the hnRNA splicing
• hnRNP-proteins participate on transport of mRNA to cytoplasm
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nnflNA ůnB-mPNAf-nnRNP/snFlNP
Aty/FIEF^
RNPSlO 0W SRm160 O
Splicing
sr>RNP
CEJC '
A/afcyre Reviews Molecular Cell Biology 3, 195-205 (March 2002)
rnRNA-rnRNP
Nature Reviews | Molecular Cell Biology
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Binding of 7-metylguanosine (m7G) via three phosphate groups to 5'-end of hnRNA by 5'-5' bound
Last two 5'-end nucleotides could be aslo methylated
m7G plays important role during initiation of translation
HO OH
v\ //
N NH,
o:= P— O
N NH,
O — CH, N
0= P—O
<1
N NH,
17
pppNpRNA
Pi
RNA Triphosphatase (RTPase)
ppNpRNA
RNA Guanylyltransferase (GTase)
GpppNpRNA
GTP PPi
SAM SAH
4
RNA (guanine-N7)-Methyltransferase (N7MTase)
m7GpppNpRNA
SAM SAH
(Cap-0 RNA)
3
RNA (nuceloside-2'-0-)-Methyltransferase (2'0MTase)
0:= p —o
m7GpppNmpRNA
(Cap-1 RNA)
DOI: 10.5772/56166
Addition of of 50 - 250 adenosines to 3'-end of hnRNA = polv(A) sequence
Catalyses by polv(A)-polvmerase
Poly(A)-polymerase is a subunit of complex, which binds on polyadenilation signal of hnRNA
Poly(A) tail is important during transport of mRNA to cytoplasm and for its stabilisation
RNA capping and polyadenylation
coding noncoding sequence sequence
Figure 7-16a Essential Cell Biology 3/e(© Garland Science 2010)
Eukaryoilc translation
> It proceeds in 2-3 compartements, cytoplasm,
> mitochondria, and chloroplasts
> The first AA is not fMet, but Met, which binds to a specific initiator tRNAjMet, which recognize the AUG codon
> The number of initiation factors which are necessary to beginning of translation is higher in eukaryotes
> The number of initiation factors which are necessary to beginning of translation is higher in eukaryotes
http://www.ncbi.nlm.nih.gov  Taxonomy     Genetic Codes
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> similar as a translation in prokaryotes
> initiation, elongation, termination
> Particular complexes are more complicated
> More of translation factors
> Genetic code of mammalian mitochondria has different meaning of some codons, 22 tRNA
> Eukaryotic cell possesses 45 tRNA with different anticodons
> Speed of translation -1-20 AA/s, depends on species and enviroment
Ferreira-Cerca, S. et al. (2007): Analysis of the In Vivo Assembly Pathway of Eukaryotic 40S Ribosomal Proteins, Molecular Cell 28, 446-457, November 2007
> Free ribosomes occur in cytoplasm
> synthesis of intracellular proteins
> the rest is bounded to the endoplasmic reticulum
> rough ER = covered by ribosomes
> smooth ER = without ribosomes
> synthesis of extracellular proteins
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> 40S subunit with bound tRNAjMet in P-site and initiation factors recognise m7G cap of mRNA
> Subsequently, this complex moves to 3-end until finds the initiation codon AUG
> Large 60S subunit binds to 40S subunit using the energy from hydrolysis of GTP
Nature Reviews Neuroscience 5, 931-942 (December 2004) doi:10.1038/nrnl557
Elongation Nature Reviews I Neuroscience
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D
Nature Reviews Molecular Cell Biology 11, 113-127 (February 2010)
doi:10.1038/nrm2838
elFJi
tRNA mRNA
elMF complex
4JS premutation complex
eRFl and eRf 3
/       1 Ribosome recycling A
ABCtl C 3
elF3< eiFIA ( elFll
5 Attachment to mRNA f>
aaa. a a
^6 5' to 1' scanning
7 Initiation codon recognition. I hydrolysis of elF2 bound GTP f   and P, release
48S initiation complex
(parriilloss)
elFS
elf}
Po« TC
i-r-rr
Elongation A
80S initiation complex
f_, 9 Hydrolysis of clFSB-bound GTP J. ĚĚlP    and release of elFSB and elFIA V
^/r^-,_-©—--p
60!
elFSB
Nature Reviews Molecular Cell
Biology
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The canonical pathway of eukaryotic translation initiation is divided into eight stages (2-9). These stages follow the recycling of post-termination complexes (post-TCs; 1) to yield separated 40S and 60S ribosomal subunits, and result in the formation of an 80S ribosomal initiation complex, in which Met-tRNAMetj is base paired with the initiation codon in the ribosomal P-site and which is competent to start the translation elongation stage. These stages are: eukaryotic initiation factor 2 (elF2)-GTP-Met-tRNAMetj ternary complex formation (2); formation of a 43S preinitiation complex comprising a 40S subunit, elF1, elF1 A, elF3, elF2-GTP-Met-tRNAMet; and probably elF5 (3); mRNA activation, during which the mRNA cap-proximal region is unwound in an ATP-dependent manner by elF4F with elF4B (4); attachment of the 43S complex to this mRNA region (5); scanning of the 5' UTR in a 5' to 3' direction by 43S complexes (6); recognition of the initiation codon and 48S initiation complex formation, which switches the scanning complex to a 'closed' conformation and leads to displacement of elF1 to allow elF5-mediated hydrolysis of elF2-bound GTP and P; release (7); joining of 60S subunits to 48S complexes and concomitant displacement of elF2-GDP and other factors (elF1, elF3, elF4B, elF4F and elF5) mediated by elF5B (8); and GTP hydrolysis by elF5B and release of elF1 A and GDP-bound elF5B from assembled elongation-competent 80S ribosomes (9). Translation is a cyclical process, in which termination follows elongation and leads to recycling (1), which generates separated ribosomal subunits. The model omits potential 'closed loop' interactions involving poly(A)-binding protein (PABP), eukaryotic release factor 3 (eRF3) and elF4F during recycling (see Supplementary information S5 (box)), and the recycling of elF2-GDP by elF2B. Whether eRF3 is still present on ribosomes at the recycling stage is unknown.
> Only one termination factor = eRF
> Disociation of ribosome from mRNA needs the energy from GTP
THE CELL, fourth edition, Figuro 10.8 O 2O0S ASM Praas and Srmm AmocMm. mc