Structure of prokaryotic genom, replication and gene expression in prokaryots
Molecular Biology
RNDr. Jan Hošek, Ph.D. hosekj @vfu Jdz
Structure of prokaryotic genom
Genom of prokaryotic cell
> no nucleus envelope
> DNA, HLP-proteins (histon-like proteins), nonhistone proteins
> nucleoid is attached to cell membrane on several
> places (Inc)
Prokaryotic chromosome
> Part of nukleoid (prokaryotic nucleus)
> Mostly circular dsDNA (linear e.g. for Borrelia burgdorferi)
>Superhelix divided to loops (domains)
Dynamic of nucleoid
a Exponential phase of growth
b Stationary phase of growth
RNA polymerase ^ at RNA promoters
iH-NS
o
Transcription factories
OFis
doi:10.1038/nrmicro2261
Nature Reviews | Microbiology
a | The folded chromosome is organized into looped domains that are negatively supercoiled during the exponential phase of growth. In this phase, the abundant nucleoid-associated proteins histone-like nucleoid-structuring protein (H-NS) and factor for inversion stimulation (Fis) bind throughout the nucleoid and are associated with the seven ribosomal RNA operons. As shown here in two cases, these are organized into superstructures called transcription factories, b | In stationary phase the rRNA operons are quiescent and Fis is almost undetectable. The chromosome has fewer looped domains, and those that are visible consist of relaxed DNA.
Plasmids
> bear genes which are not necessary for life (e.g., resistance to antibiotics)
http://www.wikiwand.com/
> circular dsDNA
> every is replicon = bears locus on
> Bears locus Inc for attachment to membrane
DNA gyrase
r
Topolsom erase
R^kHied.cawalEntly closed Circular DNA
DNA lig^sc
."v .'
linear DNA
EndonuclEaüe
Supe reo I led DMA
S
v.
, open circular DNA
supercoiled DNA
Oper clrc Lilár DMA
http://nptel.ac.in/courses/102103047/39/
EndoruclerHK-E
Replication of prokaryotic genom
transcription
DNA
translation
replication
RNA
protein
DNA
Reverse transcription
replication
RNA
Replication
DNA replication is a process in which the two strands of a DNA double helix are separated and a new complementary strand of DNA is
synthesized on each of the two parental template strands.
This mechanism ensures that genetic information will be copied faithfully at each cell division.
What is needed for DNA
replication ?
• Template strand (DNA matrix)
• Primer (free 3'-0H end)
• Polymerase + replication proteins
• dNTP
dATP + dTTP + dCTP + dGTP
Characteristic features of replication
It is semiconservative
• Meselson - Stahl experiment
experiment in biology"
jj
the most beatiful
n	i
LI	1
https://www.youtube.com/watch7v-4gdWOWjioBE
It is semidiscontinuous
...Keep a while ©
Old New strand strand
New Old strand strand
Replication of prokaryotic
genom
parental dsDNA
on
replication fork
ter
>
>
>
/
initiation elongation od DNA strand termination
Synthesis of DNA during replication
5' end of strand
r
0
o-p-o
1
0
primer strand
0
~o-p=o
1
o
h,
oh
I    3' end of strand
Vo
"()-!'-<)-!'-( - p-o-ch,
o o
L
o~ o~
i
o"
pyrophosphate
oh
incoming deoxyribonucleoside triphosphate
y end of strand
I
O
(II,
0
1
()=|'-() I
o
tlillipl.ltl!
strand
( h,
v      i 1
0
o-p-o"
1
o
https://cz.pinterest.com/pin
5' end of strand
Growing of nucleotide chain
o
nh
h,c
5-end
o
o       o o
II II II
ho—p-o-p— o— p -o—c ii
III
oh oh oh
h
oh
^ nucleosidetriphosphate
3 , 5 -phosphordiester bond
oh      3-end
\
nh
0 o o
II II II
ho—p- o—p— o— p -o
1 I I
oh oh oh
ch
Replication proteins -1
>    DNA polymerase I
> one globular polypeptide, M = 109 000
> polymerase, and 5 - 3', 3 - 5' exonuclease activities
> It catalyses replication in space between Okazaki fragments
> It removes RNA-primers by its 5 - exonuclease activity
>    DNA polymerase II
> monomeric, M = 90 000
> Polymerase activity
> 3 - 5', 5 - 3 exonuclease activity
>    DNA polymerase
> M = 900 000, oligomeric protein which consists of from several units
> It catalyse synthesis of leading strand and Okazaki fragments during replication
> It polymerize by speed 500 nt/min
> It is processive for the whole DNA molecule
Polymerase III
Dimer of polymerase III = Polin*
Poll 11* - speed 20 nt/s, procesivity 11 nt
Poll 11* + ß-clamp - speed 500 nt/s, procesivity „°°"
Sliding clamp
Clamp loader
Sliding clamp
Y-complex
Core enzyme dimerization
http:/ / slideplayer.com/slide/8771653/
LAGGING STRAND
LEADING STRAND
DPI: http://dx.doi.Org/10.1111 /i. 1574-6976.2012.00338.x
Endo- a exo-nuklease activities
5'- end
endo
3'- end
5- exo
3'- exo
-Pa-OH ends !!!
5- exonuklease activity
3- exonuklease activity
Replication proteins - II
> DNA-ligase
> It forms phosphodiester bond between 5 - and 3 - ends of two polynucleotide strands
> It joins Okazaki fragments
P liWcTdftFiWcMcTiM*!OH I.......H eTiUFiMcfcftj
OH
Replication proteins - III
> DNA-primase
> DNA-dependent RNA-polymerase
> synthesis of RNA-primer
> DNA-helicase
> untwists strands from dsDNA
> destroyes hydrogen bonds by using the energy from NTP
> DNA-gyrase (topoizomerase II)
> transform positive supercoiling to negative
Replication
replication fork
Initiation of replication
> DnaA proteins recognise the origin of replication oriC (245 nt) -» disintegration of H-bonds -» opening of oriC
> Helicases bind on released free DNA strands -> unwinding of dsDNA in the direction 5 - 3' -> creation of replication fork
> SSB-proteins bind to ssDNA parts, the proteins keep the strands in outstretched conditions; it protects reforming of dsDNA
Recognition of oríO and initiation of replication
oríC
3c 5t
r
DnaAATP
13-mef 9-mer
DNA helicase (DnaB)
DNA helicase Loader (DnaB)
3c 5«
DNA primase Q
1'
3c 5"«=
>3*
d5* ^3"
>• cn
O nO O (M CH CO
a oi "5 u_ u
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Process of replication
3 RNA-primer (11 nt),
Leading strand
imase DNA-polymerase
3'
Lagging strand
5'
5'3'
movement of replication fork
helikase
SSB-proteins
Okazaki fragments (1-2 kbp)
DNA- gyrase
DNA polymesare I removes RNA primers and synthesize the Okazaki fragments
DNA ligase joins the Okazaki fragment_
Replication is performed in
replisomes
H	
1	—i
https://www.youtube. com/watch?v=Gl AoV F3k9Hg
Pol III core
(as 9)
Two Engaged Polymerases 10 nm
B
1     I V*T
Helicase + primase = primosom
R. Reyes-Lamothe et al., Science 328,498-501 (2010)
Text to the previous picture
Fig.: Schematic model for replisome components.
• (A) Two engaged polymerases and one of the three p-clamps at a distance from the core replisome (circle of diameter 50 nm shown in gray). The data indicate that ~75% of replisomes have this organization, whereas ~25% have all three p clamps associated with the core replisome and potentially associated with active Pol III.
• (B) Expanded view of clamp loader (3') and three additional molecules of interacting with Ssb tails. The heterodimer bound to the clamp loader may also contact Ssb (14); (shown as a trimer, but the stoichiometry is unknown) then interacts with Ssb-associated.
Replication is semidiscontinual and bidirectional
Overview Origin of replication
Leading strand
Lagging strand
Lagging strand
Leading strand
Overall di of replication
Copyright O 2006 PMraon E&CMon. bx . putAtfunQ » Petno* Beo^rwi Ccrrvn-ngi
Replication - elongation
Synthesis of new DNA strands is semidiscontinuous
- leading strand
- lagging strand
- Okazaki fragments
Tsuneko Okaiaki        Reiji Okazaki
In 1968, Okazaki discovered the way in which the lagging strand of DNA is replicated via fragments, now called Okazaki fragments.
Termination of replication
Lagging strand
Replication of prokaryotic chromosome ends on specific sequences named terminators Iter)
The specific protein Tus binds to the terminators which inhibits activity b of helicase and the formation of replication fork is stopped
Replisome of E. coli and mechanism of replication fork arrest by a Tus-Ter complex.
(A) The replisome moving along the DNA template approaches Tus, and the DnaB helicase assists primase to lay down the last lagging-strand primer.
(B) DnaB helicase action isblocked by Tus, and DnaB dissociates from the template.
(C) DNA polymerase III (Pol III) holoenzyme completes leading-strand synthesis up to the Tus-Ter complex and
(D) synthesizes the last Okazaki fragment on the lagging strand, which will eventually be ligatedby DNA ligase to the penultimate fragment following removal of its RNA primer by DNA polymerase I (not shown). (E) The holoenzyme then dissociates, leaving a Y-forked structure that is single stranded on the lagging strand near the Tus-Ter complex.
,. SSB protein
DnaB helicase
Lagging strand loop
Pol III
holoenzyme
RNA primer
vO
http: / / reasonandscience. heaven forum.org/tl849p25-dna-replication- of-prokaryotes
Replication of plasmid DNA
1) Plasmids are replicon of circle type
2) They are smaller than bacterial chromosome
~~ 4 . "a
I m
o
F plasmid, periphery 31 urn
semiconservative semidiscontinuous irectional
Replication by the rolling circle mechanism
positive strand negative strand
on
rep protein
3'  New (positive) strand
Displacing strand
_ LIGATION^
SYNTHESIS OF NEGATIVE STRAND THROUGH THE OKAZAKI FRAGMENTS
Replication by the rolling circle mechanism during conjugation
Donor cell Recipient cell
The transcription of
prokaryotic genome
replication
transcription
DNA
translation
DNA
RNA
protein
Reverse transcription
replication
RNA
f
What is the transcription?
> Process of copying genetic information in DNA into RNA = synthesis of RNA from ribonucleotides on DNA strand as a template
> DNA-dependent RNA polymerase = transcriptase = prokaryotic RNA polymerase = RNA polymerase
Functions of the RNA polymerase
> It binds to promotor sequence
> It  catalyses  synthesis  of  long primary transcripts on a template DNA strand
Which primary transcripts are created during transcription?
1) Messenger RNA (mRNA)
it contains transcripts of genetic information from structural genes
2) Precursor ribosomal RNA (pre-rRNA)
primary transcript of the genes for rRNA, post-transcriptionally processed to rRNA
3) Precursor transfer RNA (pre-tRNA)
primary transcript of genes for tRNA post-transcriptionally processed to different types of tRNA
4)   Primary transcripts of regulatory RNAs
Transcription units
The transcription is performed in specific units = transcription units
1) Transcription units of non-operon type
2) Operons
Transcription units of non-operon type
starting nucleotid
Aim Aim
the last nucleotide in terminator
				
promoter	gene A	gene B	gene C	terminator
!
RNA-polymerase
transcription
primary transcript
Operon
starting nucleotid
AUG
the last nucleotide in terminator
			
promoter operator	gene A	gene B       gene C	terminator
Í
RNA-polymerase
transcription
primary transcript
!! promotor can overlap with operator!!
Types of transcription units
1)    The transcription units which contain structural genes
2)    The transcription units which contain genes for rRNA
3)    The transcription units which contain genes for tRNA
4)    The transcription units for regulatory RNA
m I
( - ^
Prokaryotic promoter
conventional sequences
starting nucleotid +1
TTGACAT
TATAAT
region -35
Pribnow box region -10
> Similarity of promoters enable their affinity to single RNA polymerase
> Differences are responsible for extent of this affinity
> strong bacterial promoter - more similar to conventional sequences
> weak bacterial promoter - less similar to conventional sequences_
Bacterial RNA polymerase
1) It is able to recognize the promoters of all transcription units
2) It consists of 5 subunits
> 2x a (M = 40 000), responsible for stability
> 1x p (155 000), used for binding ribonucleotides to the enzyme
> 1x (T (160 000), responsible for binding RNA polymerase to template DNA strand
> 1x ©, regulation
> 1 x a (85 000), a-factor, responsible for binding RNA
Q polymerase to promoter
-35 region
upstream
downstream
doi:10.3390/biom5020668
Process of transcription
Initiation of transcription
Binding of RNA polymerase to promoter of the negative DNA chain and starting of RNA chain synthesis
Elongation of RNA chain Adding of nucleoside-5 -monophosphates to 3 - end
of growing RNA strand
Termination of transcription
Stop the movement of RNA polymerase -» releasing the full length RNA -> releasing RNA polymerase from DNA
( ^ Initiation of transcription
1) Binding of RNA polymerase to the sequence -35 and to the Pribnow box (closed transcription binary complex)
2) Releasing hydrogen bonds between two DNA strands in the Pribnow box (open transcription binary complex)
3) Transcription of the first two nucleotides (open transcription ternary complex)
Negative strand
3'
a
aapp'
5'
Positive strand
Open transcription binar complex
The p subunit was removed to reveal the transcription bubble and the flipped bases in their pockets. Template DNA is in green and nontemplate DNA is in magenta, with the flipped-out bases in yellow. Bases — 11A and — 7T interact solely with the (J subunit. Base — 6G is at the (J-p subunit interface. Base +2G interacts solely with the p subunit (insert). The — 12T nontemplate base is shown in the figure as unpaired, as it is in the 4G70 coordinate set; it is likely base paired in the native promoter.
Biomolecules 2015, 5(2), 668-678; doi: 10.3390/biom5020668
Elongation of transcription
1) RNA synthesis continues in 5-3' direction and RNA polynucleotide grows
2) The speed of synthesis is about 40 nt/s
3) Once elongation starts, o-factor is released and replaced by NusA protein
3'
a-NTD
Secondary channel
RNA exit channel
RNA-DNA hybrid
Nature Reviews | Microbiology
DOI: 10.1038/nrmicro2560
aaßß'
5'
m a
Termination of transcription
1) The RNA polymerase stops its movement
2) The full length RNA is released
3) The RNA polymerase is released from DNA
4) Disociation of NusA protein from RNAP
Bacterial terminators of transcription
1. Rho-independent terminators
transcription is terminated without the presence of specific p-factor
V 1
2. Rho-dependent terminators
transcription is terminated in the presence of specific p-factor
Rho-independent terminators
3'
■ unique I sequence
JATGGTCGGGCGGATTCCTCGCCCGAAAAAAAA 5f
mRNA
5'
Hairpin with loop
'A
Negative DNA strand
AU A
UUUUUUUU3'
Rosypal, 1999
Sequence necessary for RNA release
Termination of transcription p-factor independent
Hairpin binds to NusA protein
Stop of RNAP movement
Finishing of 8U sequence transcription
RNAP releases from DNA and again associate with a-factor
terminating hairpin
NusA
RNA polymerase
DNA
Wiki commons; © Oalnefol
Rho-dependent terminators
1) Similar structure
2) The sequence AAAAAAA is replaced by another, for example GTTAGAA
3) This sequence is transcribed to CAAUCUU
4) No sequence UUUUUUUU -> no signal for RNA polymerase releasing -» dependency on p-factor
Termination of transcription p-factor dependent
• p-factor binds to rut locus of nascent RNA
• Subsequently moves towards RNAP
• Once p-factor reaches a RNAP, RNAP disociates from DNA
Rho-dependent terminator
Transcription of the structural genes
1) They contain a leader sequence (only in structural genes)
- the sequence lies between promoter and the first structural gene
- in operone it is immediately after operator
2) The leader seguence contains
- Shine-Dalqarno sequence 5'AGGA 3'
- it binds to the 3 - end of 16S-rRNA
promoter operator structural gene 1   structural gene 2 terminatoi
Transcription unit of structural genes
DNA
Prynoftr     O^fly 1 ! ^W? Zl
Shine-Dalgamo sequence |       j,^^^   Sh.ne-Dalgarno sequence
mRNA AUG x^auG
I
UG
protein
B.
protein
AAAAAA3
http://www.tankonyvtar.hu/en/tartalom/tamop425/0011_1 A_Proteinbiotech_en_book/ch01 .html
mRNA
1) Primary transcript of transcription unit bearing structural genes, which are translated into polypeptide chains
2) Contains leader sequence on the 5-end
3) Bears UUUUUUUU (ending sequence) on 3-end
4) Contains transcripts of several genes = polycistronic (polygenic) mRNA
5) Any post-transcription processing
6) Short half-life, digestion by ribonucleases in the direction 5-3'
7) mRNAs represent only 3 % of total RNA in prokaryotic cell every time
Coupled of transcription with translation = coupled synthesis
1) Ribosomes bind to mRNA during transcription
2) Both process on the same mRNA (transcription + translation)
3) In some transcription units up to 15 initiations per minute = 15 new mRNA molecules
4) On each mRNA up to 30 ribosomes = 30 new polypeptide
chains
chromosome
polyribosome
mRNA
Coupled syntheses
Influences speed of proteosynthesis
Efficient binding and progression of ribosomes along mRNA increase the speed of RNA polymerase, whereas the absence of ribosomes allows the polymerase to slow
and wait for ribosomes to catch up.
J. W. Roberts Science 328,436-437(2010)
Waaa
Coupled syntheses
1) The first ribosome translating a mRNA associates with RNA polymerase through the NusE-NusG-polymerase interaction
2) This prevents retraction of the emerging mRNA into RNA
polymerase, and thus inhibits backtracking-associated pauses that slow RNA polymerase in the absence of the ribosome.
Transcription the genes for rRNA
promoters
P1 P2
16S-rRNA
tRNA
terminators
23S-rRNA
5S-rRNA
T1 T2
M
transcription
30S pre-rRNA
posttranscriptional
processing (releasing functional products by RNase III)
23S-rRNA
5S-rRNA
Transkripce genů pro tRNA
promoter
P1	tRNA		tRNA		tRNA		EF-Tu	
DNA
transcription
v
v
tRNA
tRNA
pre-tRNA
posttranscriptional
processing (releasing functional products)
Translation of prokaryotic genom
transcription
DNA
translation
replication
DNA
RNA
protein
reverse transcription
replication
RNA
Definition of translation
> Protein synthesis
> Synthesis of polypeptide chain according the genetic code of mRNA on ribosomes
> The final process of gene (genetic information) expression
Participants of translation
> 22 activated standard amino acids
> aminoacyl-tRNA-syntetases
> tRNA
The phases of translation
> Activation of AA = amino acylation, charging
> Process in which AA is attached to the specific tRNA
> Iniciation
> A sequence of processes which produce initiation complex = ribosome 70S, mRNA, initiator tRNA, initiation factors
> Elongation
> The addition of AAs to the growing polypeptide chain, elongation factors
> Termination
> Finishing the synthesis on stop codon, releasing polypeptide from ribosome, termination factors
The primary structure of tRNA
> length about 74 - 95 nucleotides
> molecular mass 80 000
> 3- end = 5 - CCA - 3'
> integral part of the sequence are modified bases
>they originate by enzyme modification after transcription
> marking tRNAAla, tRNALeu,...
> Ala ~ tRNAAla, Leu ~ tRNALeu
The secondary structure of tRNA
cloverleaf
Ester bond
JL-k II
EfcJO—C-i-CHR-Amino acid
Cat "-----J
nh;
pseudouridin arm
P]'C arm
acceptor arm
dihydrouridin arm
adjacent arm
Intramolecular base-pairing -
D arm
anticodon arm
AnticodDn
G-C-C
Anticodon arm
The tertiary structure of tRNA
Activation of amino acids
1. R
ATP =*
NH, ■ »■ ■ 3
Macroenergetic bound 33QAMP
aminoacyladenylate
2. aa ~ AMP + tRNA -> aa ~ tRNA + AMP
catalysed by aminoacyl-tRNA
synthetase
Activation of amino acids
http: / / slideplayer. com / slide 78284821/
Structure of aa~tRNA
Anticodon arm
©
( ^ Aminoacyl-tRNA-syntetases
> molecular mass = 40 000 -100 000
> low homology in primary structure
> several conservative sequences
> each tRNA is specific for only one AA
> binding site for AA
> binding site for tRNA
> binding site for tRNA is able to bind similar tRNA!
> binding site for ATP
From what coming triplex CCA ?
> This triplex of nucleotides is present at 3 - end of each tRNA
> It is added by nucleotidyltransferase during posttranscriptional processing of theprimary transcript (without any template!)
> Two groups of nucleotidyltransferase exist > group I = Archae
>group II = prokaryotes and eukaryotes
> How the C is added is known for many years
> How the A is added was described in 2010 -crystalography studies by Pan et al.
B. Parietal., Science 330,937-940(2010)
From what coming triplex CCA ?
The COOH group from the asparagine acid in position 110 is used as a common base
More information in paper B. Pan et al., Science 330, 937-940 (2010)
Prokaryotic ribosomes: 70S - 30S a 50S
Binding sites on ribosome
3'
growing peptide chain
> binding site for mRNA
> aminoacyl site (A site)
> peptidyl site (P site)
> exit site for deaminoacylated tRNA (E-site)
^ > binding sites for initiation and elongation factors
The Nobel prize for chemistry 2009
For research of structure and function of ribosomes
1950 -1999
f
How the translation begins ?
1) Prominence of the codon AUG
- it codes the first AA in polypeptide chain
- it is present also inside of polypeptide chain
2) The codon AUG codes for methionine, nevertheless formylmethionine is at the beginning of polypeptide chain
- two tRNA for methionine exist - tRNAMet and tRNA*Met
Formylmetionine
H H H
I I I
H2 N —C —COOH 0=C —N —C —COOH
I
CH2 H CH2
deformylase
I
CH2      transformylase CH2
S I
CH3 CH
Methionin is formylated on Met ~ tRNAfMet
The codon 5 - AUG - 3
1) If it is at the beginning it binds Met~tRNAfMet, which is formylated for fMet~tRNAfMet, the initiation factor IF2 attends in the process
2) If it is inside, it binds Met~tRNAMet, the elongation factor EF-Tu attends in the process
Initiation of translation (prokaryotic)
fMet~tRNAfMet
binary complex
GTP
ternary complex
preinitiation complex (binding of tRNAj to P-site)
initiation complex
Initiation of translation
Shine-Dalgarno sequence
Start codon
mRNA
5'—pkUCU AG UAAGGAGGUUGLl|
U U
i—3'
/
IF3
Qlart XI
Shine- Start Dalgarno codon sequence
30S subunit
Portion of 16S RNA
I
IF3 promotes the binding of mRNA to the 30S subunit. The Shine-Dalgarno sequence is complementary to a portion of the 16S rRNA.
JJ
3'
IF2 promotes the binding of the initiator tRNA.
auuccuccacua
16S rRNA
IF2 and IF3 are released. The 50S subunit associates.
http:/ / slideplayer.com/ slide 7352485 8/
Ribosomes bind also on intergenes sequences
3'
Promoter
Gene 1
Start codon
AUG
I I
Ribosome binding site (RBS)
Gene 2
Transcription
Gene 3 Terminator
5' Template DNA strand
Start codon UAA AUG
_JT-UEi I I
|J_1S^_LJ
Start codon UAG AUG
T V
Stop RBS codon
Translation
Stop RBS codon
UAA
0
3' mRNA
Stop Untranslated codon mRNA
Polypeptide 1
Polypeptide 2   Polypeptide 3
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings.
Elongation of translation (prokaryotic) -Peptide growths N-end -► C-end
GDP + P
aa~tRNA
GTP -> GDP + P
TRANSLOCATION
6
EF-Tu
GTP
GTP
binary complex
ternary complex
TERMINATION
RF1
RF3
RF2
Elongation of polypeptide chain
Initiation
f Met (or peptide)
Termination
Discharged tRNA
O An aminoacyl tRNA binds to the A site, escorted by EF-Tu bound to GTP. During tRNA binding, the GTP is hydrolyzed and EF-Tu is released. EF-Ts helps recycle the EF-Tu.
EF-Tu j
Speed of elongation for E. coli 10-20 AA/s
O The mRNA advances by three nucleotides, the peptldyl tRNA moves from the A site to the P site, and the empty tRNA moves from the P site to the E site, accompanied by the hydrolysis of GTP bound to EF-G.
NE? (™)
Recycling of EF-Tu with help of EF-Ts
5,{UUU\
Peptide bond formation
Q A peptide bond is formed between the carboxyl group of fMet (or. in later cycles, of the terminal amino acid) at the P site and the amino group of the newly arrived amino acid at the A site.
0 2012 Pearson Education. Inc.
Wobble base paring In tRNA
• 22 AA - 64 triplets - 40 tRNAs
oooo
Phenylalanine
• Some tRNAs recognise more codons
• F. Crick proposed the wobble hypothesis in 1966 to explain the pattern of degeneracy
• 1st two bases of the codon-anticodon (5-XXo-3) pair strictly by watson-Crick rules
• The 3rd (5'-ooX-3') can wobble and this movement allows alternative H-bonding between bases to form
^ non-Watson-Crick base paring_
/tRNAs charged with the same AA, but recognise multiple codons are termed isoacceptor tRNAs
Wobble position
:
3'
http:/ / slideplayer.com/slide/3524858/
Revised wobble rules
Nucleotide of anticodon
G C A U I
xmVu xm5U5 Urn xm5U
xo5U k2C
Third nucleotide of codon
C.U G
U. C. (A), G A. U, G. (C) U. C. A
A. (G)
U.A. G A
The central role of GTPase
> GTPase is involved in the process
> EF-Tu is a part of GTPase
> A domain which binds GTP is evolutionary highly conservative and is present from bacteria to higher eukaryotes
> Hydrolysis of GTP is associated with conformation change, in which A2662 from 23S-rRNA is involved
Chemism of GTP hydrolysis?
> His84 acts as a general base
> which activate the catalytic water molecule by removing a proton
> the proton attacks y-phosphate of GTP
> GDP is released
GDPCP
" OgJm o
Switch I
R. M. Voorhees et al., Science 330,835-838(2010)
haaa
Termination of translation
> the presence of nonsense codon
> the presence of releasing factors RF1 (for UAG and UAA), RF2 (for UGA and UAA) and RF3 (stimulates the effect of RF1 and RF2)
> tRNA releases from carboxy end of polypeptide chain, and growing of this chain stops
> Polypeptide chain and ribosome are released, the ribosome divides to its subunits
Translation - video
Initiation
https:/ / www.youtube. com / watch? v=sr Y4d Jzh8
Termination
Elongation
https://www.youtube. com / watch? v=PpAg2 K 7ID4
https:/ /www.youtube. com/watch?v=MNMc 28EEkK0
The catalytic site for peptide
bond formation
P-loop (2246-2259)
B
A-loop (2547-2561)
puromycin
> P-loop and A-loop are the parts of 23S-rRNA
> No protein in the distance 18A from the catalytic site Q > crucial is nitrogen N3 on adenine A2451
The mechanism of peptide bond
formation
1 2451rt
13
aa-tRNA
Good nucleophile t
aa-tENA
IKK A
íj
1 a.
11
tRXA
Z+31
H
tXNA
pK^-2 A
Peptirlyl transfer product with new amide bond formed
H
aa-tRNA—<
Z4M
The mechanism of peptide bond
formation
"D
0 "D
H
aa~tRNA— N
H
pKa = 7,6
H
2451
\ /
\
c=o
tRNA
0)
aa~tRNA
2451
A
C=0
tRNA
H
0)
aa~tRNA
2451
H tRNA I
A+
aa~tRNA
pKa = -2
H
N
peptide \
C= O
H
H
2451
r
\
tRNA
The mechanism of peptide bond
formation
aa~tRNA
peptide \
C= 0 H
N
\
H peptide
A" aa~tRNA \__
2451A H — 0 v       -c= O
N pKa = -2 \N
tRNA K y
H H
Posttranslation pročešeš
• Cotranslation modifications:
• Deformylation
• Cutting of AA from N-end
• Chemical modification of AA
• Creation of disulfidic bridges
• Glycosilation
• Formation of secondary and tertialy structure
• Posttranslation modifications:
• Peptides cut off
• Formation of quarternary structure
• Binding of prostetic groups
• Formation of supramolecular complexes