Structure of eukaryotic genome, replication and gene expression in eukaryotes
Molecular Biology Brázdová Renčiuk
MB-4-2022
1
1. Structure of eukaryotic genome
>Eukaryotic cells:
>cell wall consists of cellulose (plants) or chitin (fungi), animal cells have no cell wall
Mhey contain organelles (mitochondria, plastids)
>nucleus is divided by mitosis
>nucleus consists of chromatin:
>dsDNA > histories
>non-histone proteins
>chromosomes contain linear dsDNA
MB-4-2022
2
Mitochondria
Microfilaments
Lysoso
Centrioles
>Eukaryotic cells:
reproduction: asexual (unicellular organisms), sexual
replication, transcription Microtubules and translation are more smooth complicated processes than in prokaryotes
>genes usually contain introns
Rough Endoplasmic Reticulum
Peroxisome
Nucleus
Endoplasmic Reticulum
Golgi Apparatus
Nuclear Pores
-Plasma Membrane
Nucleolus
Nuclear Envelope
Chromatin
Rough Endoplasmic Reticulum
Smooth Endoplasmic Reticulum
Ribosomes
http://pulpbits.net/7-eukaryotic-cell-structure/structures-of-eukaryotic-cells/ MB-4-2022
>Genome of eukaryotic cells: >animal cells: nucleus and mitochondria >plant cells: nucleus, mitochondria a plastids
>chromosomal (nuclear) DNA (nDNA) >mitochondrial DNA (mtDNA) >chloroplast DNA (ctDNA) >plasmids
MB-4-2022 4
Nuclear membrane
> Chromatin
> material which forms the nucleus
> consists of DNA and two types of proteins: histones and nonhistone proteins
> level of chromatin condensation is dependent on cell cycle phase
> depending on the level of condensation and the ability to be stained by basic dyes, we distinguish two types of chromatin: euchromatin (weakly stainable, decondensed, transcriptionally active) and heterochromatin (strongly stainable, condensed, transcriptionally inactive)
Chromatin fiber
c;
Chromatin fiber
A Histone and I i nonhistone proteins ^
Nuclear pore
Condensed fiber (30 nm diam.)
Histone octamer
• -N Nucleosome (11 nm diam.)
DNA
(2 nm diam.)
https://wwwxliffsnotesxom/study-guides/biology/biochemistry-ii/eukaryotic-genes/structure-of-chromatin
MB-4-2022
5
>Heterochromatin:
>Constitutive
>constantly in heterochromatin state
>centromeres, telomeres
>one X chromosome in women
> Facultative
>switches between heterochromatin and euchromatin states during ontogenetic development
>Chromatin components:
>Histones:
> small basic proteins (positively charged) which bind to negatively charged DNA
>5 types: H1, H2A, H2B, H3, H4
> high content of arginine and histidine
>Non-histone proteins:
> RNA polymerases and other enzymes of the transcriptional apparatus
>HMG1 and HMG2 (high mobility group proteins)
>HMG3 and HMG4 bind to the histone core especially in transcriptionally active regions
http://biology.kenyon.edu/courses/biolll4/Chap01/chrom_struct.html
MB-4-2022
7
>Nucleosome:
>basic unit of chromatin
>histone octamer (H2A, H2B, H3,H4)2
>1 molecule of histone H1
>DNA segment of 200 bp, which is wound around the histone octamer twice
>nucleosome fibre (10 nm) is visible by EM
octamer of core histories:
H2A, H2B, H3, H4 (each one x2) \ core DNA
Stryer, Lubert (1995). Biochemistry (fourth ed.). New York - Basingstoke: W. H. Freeman and Company. ISBN 978-0716720096
MB-4-2022
8
Discovery of nucleosome stacking in 2014
Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 2014 Apr 25;344(6182):376-80. doi: 10.1126/science.1251413.
>Chromatin domains:
>loops of 30nm chromatin fibre attached to protein scaffold (60-150 kbp)
>one molecule of topoisomerase II in the base of each loop - change of topology during replication and transcription
>each domain has one ori locus
>one human chromosome contains approximately 2000 domains
protein scaffold connecting region
MB-4-2022 10
> Mitotic chromosomes:
>originate by condensation of 30 nm chromatin fibres
Mormed during mitosis or meiosis
condensation of 30 nm chromatin fibres into 600 - 700 nm fibres, which form the chromosome structure
>in chromosomes, chromatin is in highly condensed state and is transcriptionally inactive
MB-4-2022
11
>Mitotic chromosomes:
Two chromatids Chromosome as seen via an electron micrograph
MB-4-2022 12 http://ib.bioninjaxom.au/standard-level/topic-l-cell-biology/16-cell-division/dna-supercoiling.html
>Chromosomal (nuclear) DNA: > 1 linear molecule of dsDNA
> the number of bp per one chromosome in haploid cell is 1,34x 107 to1,5x1010
> only 1.5 % of mammal genome contains protein-coding genetic information
> most structural genes are about 1 x 104 to 2 x 106 bp long - considerable part consists of regulatory
elements
MB-4-2022
13
>Repeats in nuclear DNA:
>short tandem repeats- not in prokaryotes >dispersed repeats- not in prokaryotes
>25-50% of structural genes are unique sequences
Mhe rest occurs as gene repeats
MB-4-2022
14
>Gene repeats:
>Gene family: group of genes with similar sequence, from the same original gene, generally with similar functions (genes for hemoglobin subunits) or pseudogenes (dysfunctional genes)
>Tandem gene repeats: directly adjacent, separated by spacers (intergenic sequence), genes for 5S-rRNA, genes for tRNA, genes for histones
^Dispersed gene repeats: copies are dispersed in different locations in the genome (genes for tRNA, snRNA, etc.)
MB-4-2022
15
>Chromatin organisation in the nucleus - fractal globules
2. DNA replication in eukaryotes
replication of mitochondrial and chloroplast DNA replication of nuclear chromosomes:
>semiconservative (each new dsDNA contains one parental and one newly synthesised strand) and semidiscontinuous (leading and lagging strands)
initiation, elongation, termination
>only in S-phase of cell cycle
Genetic material duplicates.
Int. xsgri-
Cell grows.
-9,%.. G2
Cell prepares to divide.
MB-4-2022 17
>Replication of nuclear chromosomes:
>proceeds in several places at once (in contrast to prokaryotes) >chomosome is a set of replicons, many ori sites (30-50k in mam >euchromatin is replicated earlier than heterochromatin
_ Incomplete ends 20-
http://www.biology-pages.info/T/Telomeres.html
>DNA replication process:
MB-4-2022
19
>Eukaryotic DNA polymerases:
>DNA polymerase a- in complex with primase synthesizes Okazaki fragments, does not possess 3'-5' exonuclease activity (no proofreading activity)
>DNA polymerase ß- synthesizes short fragments during DNA repair
>DNA polymerase y- synthesis of mitochondrial DNA
>DNA polymerase 5- synthesis of leading strand and completing the lagging strand
>DNA polymerase z- leading strand synthesis and DNA repair
MB-4-2022 20
>Eukaryotic replication fork:
Fork movement
Origin of replication
Leading Lagging   3" NT,
strand strand
Fork movement
T5'
I I I I I I 1 y
https://biology.stackexchangexom/questions/31585/does-dna-polymerase-always-go-the-same-direction
lagging strand
primase
Ccd45
SINS
Mcm2-7 hclicasc
leading strand
PCNA
MB-4-2022
21
>Histone partitioning:
>H3-H4 dimers are formed by parental or newly synthesized histone monomers
>newly synthesized dimers are associated together or mixed with parental dimers
>H2A-H2B dimers are added and nucleosome is formed
1	\		
2		1-»- €  1 ^ •	
	1 _____ Two H2A-H2B dimers ^-^^^^		
-2022 "ucleosome		22	
D. Ray-Gallet et al., Science 328,56-57 (2010)
>Nucleosome assembling is associated with hydrolysis of ATP
SWR1 -mediated histone replacement
Nuc.eosome ADp H2A2.H2B
dimer
H2A-H2B dimer
E. Luk et al.. Cell 143.725-736. Nov 24.2010
MB-4-2022
23
Origin
>Replication of linear molecules:
Mhe end replication problem
Melomerase = ribonucleoprotein - RNA is a template, protein has catalytical function
O DNA replication is initiated at the origin; the replication bubble grows as the two replication forks move in opposite directions.
Finally only one primer (red) remains on each daughter DNA molecule.
0 The last primers are removed by a 5'—•• 3' exonuclease, but no DNA polymerase can fill the resulting gaps because there is no 3' OH available to which a nucleotide can be added.
O Each round of replication generates shorter and shorter DNA molecules.
Leading strand
Lagging strand
3'
5'
3'
Gap
5'
3'
5' 3'
5" 3"
I
I
— 5
— 3
Gap
■ 3' 5'
■ 5'
■ 3'
■ 5'
iiniiriniiinWi^aV"'"'
https://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-19/CB19.html
■5'
■3'
> Telomeres:
>ends of eukaryotic chromosomes
>repeats of TTAGGG/CCCTAA sequence (in vertebrates), several thousands of repeats
>single-stranded overhang of 50 - 200 nucleotides
>protection of chromosomes against degradation (by exonucleases) or fusions
Melomeres associate with nuclear membrane
MB-4-2022
25
>Filling of missing 3'-ends:
>telomerase elongates 3'-ends
>formation of hairpin and RNA primer
>replication of complementary strand and removing of hairpin
PROCESS: TELOMERE REPLICATION
III""
Missing DNA on 5   lagging strand
T   T  T AGOOT  TAOOQT TAQQQ
1. End is unreplicated.
3
Telomerase with its own RNA template
jHflJiik O Q O
5 3
^CIC.C
TTAQGCT  TA o'c'o
2. Telomerase extends unreplicated end.
n-onnns on
IHIJO GTTAOGGTTACGG
C|A|A,U C.C,
3. Again, telomerase extends unreplicated end.
RNA primer
A A U C C C
5
IMliillMllU
4. Lagging strand is completed.
Sliding clamp
http://masteringyourwaytomedschool.blogspot.ez/p/bio-1000-dna-shortening.html
MB-4-2022
26
>Sequences of telomeres:
TABLE 11.5		
Telomeric Repeat Sequences Within Selected Organisms		
Group	Examples	Telomeric Repeat Sequence
Mammals	Humans	TTAGGG
Slime molds	Physarum. Didymium	TTAGGG
	Dictyostelium	AG(1-8)
Filamentous fungi	Neurospora	TTAGGG
Budding yeast	Saccharomyces cerevisiae TG{1_3)	
Ciliates	Tetrahymena	TTGGGG
	Paramecium	TTGGG07G)
	Euplotes	N TTGGGG
Higher plants	Arabidopsis	TTTAGGG
http://reasonandscience.he9^n|p^^.org/t2263-the-telomerase-enzyme
27
>Protection of chromosome ends:
Double-strand break
Activates DNA damage response pathways
Cell cycle arrest
ATM kinase ATR kinase
DNA repair
Homology-directed repair (HDR) Nonhomologous end joining (NHEJ)
Protected from DNA damage response pathways
T. De Lange, Science 326, 948-952 (2009) 3 MB-4-2022
28
>Mammalian telomeres:
Telomere t-loop
\
Strand invasion of 3' overhang
T. De Lange, Science 326, 948-952 (2009) MB-4-2022
29
>Different outcomes of end-protection problem:
Mammals
/<-^
^_„>___))
■■■■ ■■;:( )
TRF2
OTHERWISE
Ku
I NHEJ Dicen,r'c chromosomes ' and genome instability
I HDR
OTHERWISE
Terminal deletions and telomere length changes
OTHERWISE
G,/S or G2/M arrest and apoptosis/ senescence
Budding yeast
Rif 1
Cdc13
Stn1/Ten1
OTHERWISE
NHEJ   Dicentric chromosomes and genome instability
OTHERWISE
Telomere length changes
OTHERWISE G2/M arrest
OTHERWISE
?
MB-4-2022 T. De Lange, Science 326, 948-952 (2009)
30
>Termination of DNA
replication in eukaryotes:
> replication of prokaryotic chromosome ends in specific sequences -termination zones
>other factors involved in termination of DNA replication in eukaryotes -topoisomerase II participates and the process is regulated by ubiquitination
TERMINATION ZONE (TERs)
Fork fusion    .! I.
Mr
Fork pausing
Resolution
OPolu •Pok.6 OPausing element
MB-4-2022
DOI: http://dx.doi.Org/10.1016/j.molcel.2010.07.024
3. Transcription in eukaryotes
>Primary transcripts:
>precursor mRNA (pre-mRNA)
>heterogenous nuclear RNA (hnRNA)= pre-mRNA forming in nucleus >precursor ribosomal RNA (pre-rRNA) >precursor transfer RNA (pre-tRNA) >5S-rRNA
>small RNAs (snRNA, snoRNA, scRNA)
>Eukaryotic DNA-dependent RNA-polymerase:
>RNA-polymerase I, II, III
>Transcription factors
MB-4-2022
33
>Eukaryotic RNA-polymerases:
>RNA polymerase I:
>synthesis of pre-rRNA >only in nucleolus >insensitive to a-amanitin
>RNA polymerase II:
>synthesis of hnRNA and some snRNA >sensitive to a-amanitin
>RNA polymerase III:
>synthesis of pre-tRNA, 5S-rRNA and some snRNA
MB-4-2022 34
>RNA polymerase II
a, Representative regions of the cryo-EM density for EC1 with the refined model superimposed. Depicted are (from left to right) RPB1 helix al9, RPB8 strand (38, the DNA-RNA hybrid, and the active site aspartate loop with the bound catalytic metal ion A and the 3'-nucleotide of the RNA transcript, b, Ribbon model. The views correspond to the previously used 'top' and 'side' views of yeast Pol II and are related by a 90° rotation around a horizontal axis. Black spheres indicate the location of residues that are not identical between bovine and human Pol II, bovine indicated second (three out of seven residues, the remaining four are disordered). The final model lacked several short surface loops and flexible N-terminal residues.
Structure of transcribing mammalian RNA polymerase II Nature volume 529, pages 551-554 (28 January 2016)
MB-4-2022
35
>Transcription unit:
>monocistronic character
>contains:
> promoter
> leading sequence >polyadenylation signal
> terminator
Poly-A signal sequence
Enhancer Proximal (distal control elements)    control elements
Termination region
DNA
_A_Exon    Intron  Exon    Intron Exon_//_/_
Upstream IE I Downstream
Promoter j Transcription
Primary RNA Exon    Intron  Exon    Intron Exon    cAe*\tr*i 3' end
transcript       5'I^H^^^H^Zl^^^MZ  mH0f primary
RNA processing transcript
Intron RNA
Poly-A signal
Coding segment
mRNA    g     P p HI^^^^HH  M*aa aaa 3'
»-v-' Start     Stop [—y—^—«—'
5'Cap  5'UTR   codon   codon   3'UTR Poly-A
tail
http://nitro.biosci.arizona.edu/courses/EEB600Aj-g 42Q22 36 2003/lectures/lecture24/lecture24.html
>Transcription factors:
>necessary for transcription inititation
>bind to promoter sequence
>General TFs:
>present in all cells, necessary for initiation
>Basal - low activity, minimal cell requirements
>Constitutive - inrease the basal activity according to the cell type
>Special TFs:
>only in specific cells in specific time
Munction in inducible transcription
MB-4-2022
37
>Eukaryotic promoter:
> transcription by RNA polymerase II
constitutive and special
transcription factors        basal transcription factors
C
TFIID
TATAAAA
Hogness box (TATA box) ■34 to -26 bp
MB-4-2022
38
>Binding to TATA box:
TATA-box Binding Protein (IBP)
>recognised by basal transcription actor TFIID
>TBP Protein {TATA binding protein) is a part of TFIID, present in all eukaryotes
http://www.web-books.com/MoBio/Free/Ch4E.htm
MB-4-2022
39
>Transcription of hnRNA:
1. decoupling of transcription and translation
2. hnRNA is capped and methylated (binding to ribosome)
3. in the 3'-end (after stop codon) AAUAAA sequence is present, hnRNA is cleaved in this region
4. hnRNA is polyadenylated on 3'-end (stabilization in cytoplasm)
5. introns are removed and exons are connected to form mRNA
MB-4-2022
40
^Initiation of transcription:
1. binding of transcription factors to TATA box and other regulatory sequences - preinitiation complex
2. binding of RNAP II - closed initiation complex
3. phosphorylation of CTD domain of RNAP II by transcription factor TFIIH (helicase and kinase activity) - RNAP II activation and unwinding of dsDNA - open initiation complex
4. dissociation of RNAP II from other TFs (apart from TFIIF) and start of RNA synthesis
iption complex
MB-4-Z022
>Parts of eukaryotic promoter:
Activators
These proteins bind to genes at sites known as enhancers. Activators help determine which genes will be switched on, and they speed the rate of transcription.
Enhance/-
Repressors
These proteins bind to selected sets of genes at sites known as silencers. They interfere with the functioning of activators and thus slow transcription.
Coding region
Coactivators
These "adapter" molecules integrate signals from activators and perhaps repressors and relay the results to basal factors
http ://www.cbs.dtu.dk/d|yj:gu2f e^g2kbooks/dave/Lekt03 bkg .htm I
Basal transcription factors
In response to injunctions from activators, these factors position RNA polymerase at the start of the protein-coding region of a gene and send the enzyme on its way.
>Spatial assemblies during transcription:
B
(A) Linearly defined expression units in compact genomes and spatially assembled expression units in complex genomes.
(B) Association between coordinately expressed genes.
(C) Colocalization of genes at subnuclear structures, such as transcription factories.
Assembly of functional expression units
Expression unit Small genomes j
Complex genomes
Expression units
MB-4-2022
Dekker J: Science 319, 1793-1794 (2008)
Coordinated expression
On
Functional organization of the nucleus
Transcription factory
43
>Termination of transcription:
Merminator contains AATAAA sequence - polyadenylation signal
>polyadenylation signal in hnRNA is recognized by protein complex, which cleaves hnRNA 10-30 nt towards 3'- end
>RNAP II dissociates from DNA and the rest of hnRNA is degraded
a Termination at mRNA-coding genes in yeast
Recruitment of the CPF-CF complex
CTD -M^Phosphorylated Ser2 S'<
CPF-CF, y^complex CFIA\^
Elongation factors
TSS
jRNA
DNA Poly(A) signal
b Termination at ncRNA genes in yeast
Phosphorylated SerS
Recruitment of the NNS complex
DNA
Nrdl-and Nab3-binding sites
RNA cleavage and polyadenylation
Dissociation of the elongation complex
-sr..-
Allosterk model
RNA polyadenylation and degradation or processing
MB-4-2022
Nature Reviews Molecular Cell Biology\6, 190-202 (2015) Nature Reviews | Molecular Ceil Biology
44
termination of transcription:
a Termination at mRNA-coding genes in metazoans
CTD    Phosphorylated ^ *TSer2 ^
Transcription
RNA cleavage and polyadenylation
Recruitment of iCPSF-CF compl
DNA
Dissociation of the elongation complex
Torpedo model
PAS R-loop b Termination at genes encoding snRNAs in metazoans
Recruitment of termination or processing factors
"Phosphorylated Ser2 Phosphorylated Ser7
Dissociation of the elongation complex
snRNA
3' end processing       y 5
snRNA gene 3'box
Nature Reviews Molecular Cell Biology 16, 190-202 (2015)
XRN2 4^ ^>
MB-4-2022
Nature Reviews | Molecular Cell Biology
45
>Transcription and nucleoporins:
>in yeast, frequently transcribed genes are located near nuclear pores
>after activation of transcription, activated regions are transported to nuclear surface
Multicellular organisms contain lamins, which are localized in inner surface of nucleolema (not yeast)
Ikegami, K. a Lieb, J. D. Plos Genetics 6 (2), 1-2 (February 2010)
MB-4-2022
46
>Transcription and nucleoporins:
>nuclear pore complexes (NPC) selectively transmit macromolecules
>complexes of more than 400 proteins (nucleoporins) in 30 subunits
>nucleoporins Nup153 and Mtor form filamentous structures which transport DNAfrom inner part of nucleus towards nuclear pores
http://cellbio.emory.edu/lab/powers/Research.html
Ikegami, kM Bli&^B. Plos Genetics 6 (2), 1-2 (February 2010)
47
>Posttranscriptional RNA processing:
>hnRNA modifications:
>Formation of hnRNA-protein compl >Adding cap to 5'- end >Polyadenylation of 3'- end >Splicing of hnRNA
MB-4-2022
>Formation of hnRNP complexes:
>Proteins which specifically bind hnRNA- hnRNP proteins
>Proteins which specifically bind to small nuclear RNA (snRNA)- snRNP proteins
>snRNP proteins + snRNA= snRNP particles
>hnRNA+ hnRNP proteins + snRNP particles= hnRNP complex
>snRNP particles bind to introns and form spliceosome
>hnRNP proteins participate on transport of mRNAto the cytoplasm
MB-4-2022
49
>Formation of hnRNP complexes
mRNA-hnRNP
Natu« nwiwM I Moi«ui«Wßi-Äö2Ä22A\/afuA'e Reviews Molecular Cell Biology 3, 195-205 (March 2002)
>Adding cap to 5'-end of hnRNA:
>Binding of 7-methylguanosine (m7G) via three phosphate groups to 5' end of hnRNA via 5'-5' linkage
>Also two or three 5'-end nucleotides can be methylated
>m7G plays and important role in initiation of translation
! HN
1 ^ I NH • n
I
CH.t«- present in fsj v      all caps
0
II
P
1
o
NH2-
0 CpN CH2 op 0 P OP O CH?
O
NH.
G added by 5'   5' linkage
present in cap 1
(CH3 group in two terminal 5' nucleotides)
~l
this position can be methylated also in cap 1
6 OCH3
s\0" present x
in caps 2 N
(CH3 group in three
terminal 5' nucleotides) primary
transcript
H
-CH?
Ö
O OH
3'
http://www.biocydopediaxom/index/genetics/expression_of^
of_caps_m7g_fj)pg(_4^[52-2olya_for_mrna.php 52
>Polyadenylation of 3'-end:
>Addition of 150-250 adenosines to 3'-end= poly(A)sequence >Catalysed by poly(A)-polymerase
>poly(A)-polymerase is a part of complex which binds to polyadenylation signal on hnRNA
>poly(A)-end is crucial during transport of mRNAto cytoplasm and its stabilization
RNA capping and polyadenylation
coding noncoding sequence sequence
AAAAA
poly-A tail
150-250
protein
Figure 7-16a Essential Cell Biology 3/e (« Garland Science 2010)
MB-4-2022
52
>Splicing of hnRNA:
>lntrons are cleaved out of hnRNA to form mRNA > Structure of intron:
>GU-AG rule (donor and acceptor sites) > Branch site
Splice donor site
Branch site
Splice acceptor site
Exon
<20
Exon
7 AG GT £ AG
A G
I NCT_AAC ................ CCCCCCCCCCNC
CTCGT TTTTTTTTTTT Pu Py Py rich
G A
Pu
Consensus sequences at the DNA level in introns of complex eukaryotes
http://www.geneinfinity.org/sp/sp_coding.html
MB-4-2022
53
>Splicing of hnRNA:
Mransesterification - no energy from ATP or GTP is needed
>snRNA and snRNP particles play crucial role
>intron is cut out in the form of lariat RNA
precursoi 1o tiHNA
boo I
5' splice (
® a
A G
["OH MX l-OM
t«-..-;-
branch point
L
>
i. i
3' spfcce site
A G
phoapftoeeter transfet
I ? OH ol 1  a! 5 splh
branch nucleotide A attacks 5 phoaphoryl splice »w
I   <S>>        * a
y Um     oi ci
^ p • p tHH»-pJ-pJ-|>H
I     ®PG A <
0
1
3 OH ol exon 1 attacks
5' phosphoryl at 3f spice site
spfcoed exons
■on -oi
exosod larwt
Vox z^m*
S pHoshoryl p J" P 9 mnctwy riboso
MB-4-2022
http://www.bx.psu.edu/~ross/workmg/RNAProcessingChl2.htm
54
>Role of Mg2+ ions during splicing:
>Self-splicing:
>Rare autocatalytic process of hnRNA splicing
>No proteins (enzymes) are needed
>Digestion and ligation of RNAduring self-splicing is catalyzed by ribozymes (ribonucleic acid enzymes)
MB-4-2022
56
>RNA editing:
>posttranscriptional insertion or deletion of nucleotides in RNA or conversion of one base to another
>results in RNA transcript which does not correspond to original coding sequence in DNA
>Two types:
1. site-specific deamination
2. gRNA (guide RNA)-directed editing
MB-4-2022
57
>Deaminaton C -> U:
>ln specific mRNAs only in certain tissues or cell types
>Two forms of apolipoprotein B
>Liver: long Apo B-100
>lntestine: short Apo B-48
> Formation of stop codon UAA
Cytidine deaminase
MB-4-2022
>i7iRNA editing in apolipoprotein B gene:
pre-mRNA
I Qln -1 I I
Apo B-100 4 563 aa Apo B-48 2 153 aa
MB-4-2022
59
>Deamination A -> I:
>ln ion channels in mammalian brain
>Single nucleotide conversion changes the coded aminoacid
>This changes the permeability of ion channel to Mg2+ ions
adenosine    hbose-5-phosphate inosine ribose-5-phosphate
or 2'-deoxyribose-5-phosphate or 2'-deoxyribose-5-phosphate
MB-4-2022
60
>gRNA-directed editing:
>gRNAs (guide RNA) are 40-80 nucleotides long
>First described in coxllgene in Trypanosome
>Enable adding of U in specific regions of transcripts
>gRNAs bind to mRNA, enable their splicing, adding the missing nucleotides and linking of the spliced fragments
>Resulting mRNAs contain large segments of added U and lose several U from original sequence
insertions of U can be present in up to 50 % edited mRNAs
MB-4-2022
61
>gRNA structure:
>each gRNA has 3 regions:
1. first, in 5'-end (anchor), enables binding of gRNA to region of mRNA editing
2. the second directs which nucleotides will be inserted
3. polyLl sequence at 3'-end
(poly U)
region of editing
region of anchoring
MB-4-2022
62
> Sequence of gRNA and unedited mRNA
unedited RNA
GAGAACCU
r
gRNA
(polyU) CUAA CAUAUGGA
L
1
J
region of editing
region of anchoring
MB-4-2022
63
>Process of editing I:
location of U addition
mRNA
GAG   A ACC
gRNA
mRNA
gRNA
(polyU) CUAA CAUAUGGA
I_I_I
UCAUAUGG
J d
MB-J2022
igestion by endonuclease
64
>Process of editing II
Addition of dUTP
U U
mRNA
ecu
gRNA
(poly U)      CU CAUAUGGA
[ AA
ligation
mRNA
GA      G   A ACCU
gRNA (poly uTK C U AA CAUAUGGA
MB-4-2022
> Transcription:
https://www.yotA?ÍD1é^9)?ŕT/watch?v=WQbE7I-tV44
66
>Codons:
u
Second nucleotide
C A
a>
—
o o
o 3 C
U
uuu uuc
Phi
UUA UUG
cuu cue
CUA CUG
AUU AUC
He
AUA
AUG Met
GUU GUC GUA GUG
ucu ucc
UCA UCG
ecu ccc
CCA CCG
Pro
ACU ACC ACA ACG
GCU GCC GCA GCG
UAU
Tyr
UAC
UAA STOP UAG STOP
CAU CAC
His
CAA CAG
Gin
A'AJ Asn AAC U
AAA AAG
Lys
GAU GAC
GAA GAG
UGU
Cys
UGC W UGA STOP
UGG
CGU CGC CGA CGG
AGU AGC
Ser
AGA AGG
GGU GGC GGA GGG
https://www.nature.com/scitable/content/the-amino-acids-specified-by-each-mrna-6903567
MB
1-4-2022
4. Translation in eukaryotes
>Differences from prokaryotic translation:
>it takes place in 2-3 compartments - cytoplasm, mitochondria, chloroplasts
>the initial AA is not fMet but Met, which binds to a specific initiation tRNAiMet that recognizes the initiation codon AUG
>the number of initiation factors required for translation initiation is higher than in prokaryotes
MB-4-2022
68
>Translation in eukaryotes:
>Similar to translation in prokaryotes
initiation, elongation a termination
>Individual complexes are more complicated
>Higher number of initiation factors
>Genetic code in mammalian mitochondria has a different meaning for some codons, 22 mitochondrial tRNA genes
>Eukaryotic cells possess 45 tRNAs differing in anticodons
translation takes place at 1-20 AA/s, depending on organism and conditions
MB-4-2022 69
>Cytoplasmic ribosomes:
> Also involved in the formation of ribosomes:
> 150 non-ribosome proteins
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
MB-4-2022
70
>Bound and free ribosomes:
>Free ribosomes occur in the cytoplasm
intracellular protein synthesis
>Others are bound to endoplasmic reticulum (ER)
>Rough ER is covered with ribosomes
>Smooth ER is ribosome-free
Ribosome
http://www.assignmentpointxom/science/biology/about-ribosome.html
MB-4-2022
71
initiation of translation:
>40S subunit with tRNAiMet bound at the P site, initiation factors bind to the m7G mRNAcap
>The whole complex moves in the 5-3 direction before it hits the AUG initiation codon
>Hydrolysis of GTP, the 60S subunit binds
MB-4-2022
72
https://www .youtube.com/watch?v=kAeLta-BstO
MB-4-2022
73
(a) Formation of the cap-binding complex
40S
(b) Formation of the ternary translation initiation complex
GDP
Suppression of translation initiation
40S
mRNA
TRENDS in Motocuter Mk*C*w
iio- „ D
oh   no o
0=p o
An
HO OH
1 B = Ade
2 B a Gua
C
c
I
S'pA-0 G-C
6-6
G-6 6 6 G-C
I
D
.G...A
U
G.
C G C tr^G
G-O-G
m
*>4
G A^-G C C JMJ -C---G-G
• A.
fn,A
--C,
rn,C-D
'A...
u"
.0
L..A
in;G
e-Gt,. A-g A'
M
r,-G_C«i
G-C
C' 'A
m7G
LI
c...A.u
MB-4-2022 74
http://mol-biol4masters.masters.grkraj.org/html/Protein_Synthesis4-Eukaryotic_Mech
tRNA carrying first amino acid
Ribosomal subunits
UAC Anticodon
mRNA
AUG Start codon
UAG Stop codon
INITIATION
5'
			JU&OUUUU"_"JUUULTLJUlJUUlJUl^	
				
O During initiation, the components ^ of the translational apparatus come \ together with an mRNA, and a tRNA ', carrying the first amino acid (AA,) binds to the start codon (AUG).
ELONGATION
Q During elongation, amino acids are brought to the mRNA by tRNAs and are added, one by one, to a growing polypeptide chain.
5' ruULTULJUl
© 2012 Pearson Education. Irw.
~~UAG Stop codon
https://www.munxa/biology/dM^l7fri^h9^L2060/BIOL2060-22/22_07.,
0 During termination, a stop codon in the mRNA is recognized by a protein release factor, and the translational apparatus comes apart, releasing a completed polypeptide.
75
>Termination of translation:
>Eukaryotic translation termination factor eRF1 (release factor) recognizes all 3 stop codons
https://www70utubexofn/^vatcrT)^C4QiMqBSDe4
>Extracellular and membrane proteins:
>all extracellular and membrane proteins have a 15-25 AA signal peptide at the N-terminus
>signal peptide binds to the signal recognition particle (SRP)
>SRP stops translation on the ribosome
>binding of SRP to the receptor on the membrane leads to cleavage of the signal peptide by signal peptidase and the translation continues
MB-4-2022
77
>Extracellular and membrane proteins:
membrane
MB-4-2022
78
>Translocation of extracellular protei
THE CELL. Fourth EditKi*. Figur* 10.8 O 3O06 ASM Prws wx) Sinfertr AMOdMS. UK.
The Cell, Fourt edition, Figure 10.8. 2006
MB-4-2022
>Formation of membrane proteins
TH£ CCU, Fourth foVTwi. Ftguro 10.12 O 2005 ASM Prws arxj S*Vfc*r AsWCWM. lot
The Cell, Fourth edition, 06
80
>Translation and folding:
>ln some proteins, the ribosome shift briefly stops following synthesis of short oligopeptide
>This will allow precise targeting of the protein to the exit tunnel in the large subunit
>Precise targeting of the protein to the tunnel is associated with proper folding
r-iRYA
Ribosome
Large subunit Small subunit
MB-4-2022
Peptidyl
Transferase
Center
Decoding Center
http://www.crcl.fr/311-Projets-en-GB.crcl.aspx?lang uage=en-(5B
MB-4-2022
82