Replication and gene expression in
prokaryotes
Brázdová
Renčiuk
MB-3-2022 1
Structure of prokaryotic genome
NUCLEOID PLASMIDS
• Not designated by a nuclear membrane
• DNA, HLP (histone-like proteins), non-histone
proteins
• Prokaryotic chromosome - DNA is one circular
molecule of dsDNA
• E. coli – 4,7 Mbp - >4000 genes
• Attached to cellular membrane in several places
• Origin of replication oriC and termination region ter
• Small circular molecules of dsDNA (1-200 kbp) – up
to several thousands per cell (usually 100s)
• They carry genes that are essential for survival
(resistence to antibiotics e.g.)
• Every plasmid is a replicon (has its own oriC) and
replicates independently of the genomic DNA
• Bacteria can acquire plasmids by conjugation or
from the environment
• Great importance in biotechnology
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Prokaryotic chromosome
• Circular molecule
• Organized into looped domains
• Each loop is independently supercoiled
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DNA replication
• Synthesis of new DNA molecule according to template
• Rules of base complementarity
• Key element of cell division
DNA
RNA
Protein
Transcription
Translation
Reverse transcription (retroviruses, …)
Nucleic
acids
Replication
Replication
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Replication goes in both directions
• Bacterial chromosome has one origin of
replication (oriC)
• Replication goes in both directions from
oriC
• Replication ends in ter region
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Replication fork
https://courses.lumenlearning.com/microbiology/chapter/dna-replication/
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DNA replication rules
• Template - DNA strand used for synthesis
• Primer – free 3‘OH end
• Replication proteins – DNA polymerase, primase
• dNTP – deoxyribonucleosidtriphosphates (dATP, dGTP, dCTP,
dTTP)
5‘ 3‘
-OH
5‘3‘
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DNA replication is 5‘-3‘ directed
• Strand growth can occur in both directions - 5‘-3‘ or
3‘-5‘ from template
• 3‘-5‘ – different polymerase (artificial)
• Problem is in the proofreading mechanism – repair
of wrongly incorporated base: in the case of 3‘-5‘
polymerization – termination of replication
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DNA replication is SEMI-DISCONTINUOUS
• Replication proceeds on both strands simultaneously – template strands have
opposite polarity (5‘-3‘ and 3‘-5‘) – they are antiparallel:
• leading strand - continuous synthesis from one primer
• lagging strand - discontinuous, Okazaki fragments from multiple different primers
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DNA replication is SEMI-CONSERVATIVE
• New molecule of dsDNA contains one strand from template DNA and one strand
of newly synthesised DNA
• Meselson-Stahl experiment – 15N labeled DNA in bacteria further cultivated in 14N
environment
• www.youtube.com/watch?v=JcUQ_TZCG0w
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DNA synthesis during replication
N
O
O
O
P
OH
O
OH
N
O
O
O
P
O
OH
N
O
O
OH
P
O
OH
N
NH2
O
N
NH
N
NH2
O
NH
O
O
CH3
N
O
O
OH
P
O
OH
P
O
O
OH
OH
OH
O
O
P N
N
N
NH2
P
O
O
OH
OH
OH
OH
O
P
Phosphodiester
bond
5‘ end
(phosphate)
3‘ end
(hydroxyl)
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Replication proteins - polymerases
{Fijalkowska J. et al., FEMS Microbiology Reviews, 2012}
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Replication proteins - polymerases
Processivity
• Ability to synthesize the reaction without releasing the template – in case of DNA synthesis it is incorporation of
nucleotides – measured in nucleotides, without dissiciation of polymerase - high processivity (leading strand
synthesis), low (synthesis of spacers between Okazaki fragments)
Fidelity
• Ability of DNA polymerase to copy template strand, how it incorporates complementary bases – relates to
polymerase errors (e.g. 105 correctly incorporated nucleotides to 1 error) - higher because of proofreading activity
of polymerase = ability to cut out incorrectly incorporated nucleotide (3‘-5‘ exonuclease activity)
Speed
• Average amount of nucleotides incorporated per second – connected to processivity
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Replication proteins - polymerases
DNA polymerase I
• Globular protein; Mw = 109 000
• Multidomain structure – own polymerase activity, 3‘-5‘ exonuclease (proofreading) and 5‘-3‘ exonuclease activity
• Replication in spaces between Okazaki fragments
• 5‘-3‘ exonuclease activity ensures degradation of RNA primers during ligation of Okazaki fragments
• Low processivity (20-25 nt), low synthesis speed (10-20 nt/s), high fidelity (104-105)
DNA polymerase II
• Monomer; Mw = 90 000
• Polymerase and 3‘-5‘ exonuclease (proofreading) activity
• DNA repair
DNA polymerase III
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Polymerase III
• Complex of several proteins ; Mw = 900 000
• Basic complex of subunits α, ε a θ, through τ subunits form a dimer,
further associates with subunits γ and β clamps which raise the
processivity of enzyme
• Replication of leading and lagging strand
• Processivity and speed of synthesis depends on the structure of
complex (monomer x dimer; presence of β clamp) – whole complex
syntesizes DNA 1000 nt/s with high processivity
• Polymerase and 3‘-5‘ exonuclease (proofreading) activity
{Fijalkowska J. et al., FEMS Microbiology Reviews, 2012}
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Exo- and endo-nuclease activity
DNA5´ 3´
5´-3´ exo 3´-5´exo
endo
N
O
O
O
P
OH
O
OH
N
O
O
O
P
O
OH
N
O
O
OH
P
O
OH
N
NH2
O
N
NH
N
NH2
O
NH
O
O
CH3
5‘ end
(phosphate)
3‘ end
(hydroxyl)
5´-3´ exonuclease
3´-5´ exonuclease
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Replication proteins - other
DNA ligase
• Creates phosphodiester bond between 5‘ end and 3‘ end of two polynucleotide strands
• Connects Okazaki fragments
DNA primase
• DNA-dependent RNA-polymerase
• Syntesizes RNA primers (one for leading strand and one for each Okazaki fragment)
DNA helicase
• Unwinds DNA strands in duplex
DNA gyrase (topoisomerase II)
• Unwinds superhelical twists created by replication fork progression
• Converts positive superhelicity to negative
Replication proteins form complex – replisome
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Replication progress
• initiation: requires primer in replication origin: short RNA synthesized by
primase
• elongation: DNA polymerase creates new strand in 5'—>3' direction
• termination: in ter region
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Replication initiation
1. Recognition of replication origin (oriC) by DnaA proteins, relaxation of
hydrogen bonds at oriC
2. Binding of helicase to unwound DNA strands – unwinding of duplex DNA in
5‘-3‘ direction – creates replication fork
3. SSB proteins bind to ssDNA (single-strand binding), they keep DNA in
unwound state
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Replication progress
• Unwinding of dsDNA by helicase
• Relaxation of superhelicity by topoisomerase
• Binding of SSB proteins to ssDNA
• Synthesis of RNA primers on the lagging
strands in Okazaki fragments by RNA primase
• Synthesis of DNA on leading and lagging
strands by DNA polymerase III
• Cleaving of RNA and synthesis of spacers in
Okazaki fragments by DNA polymerase I
• Ligation of DNA segments on lagging strand by
DNA ligase
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Replication termination
• Termination region = ter
• Tus protein binds to ter
• Tus – inhibits the activity of helicases (DnaB
proteins)
http://reasonandscience.heavenforum.org/t1849p25-dna-replication-of-prokaryotes
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Replication of plasmid DNA
• Plasmids are circular molecules of double-stranded DNA
• Plasmid = circular replicon (origin of replication)
• Replication by rolling circle mechanism – can result in
product containing multiple copies of DNA – concatemer
• Smaller plasmids are replicated by host cell replication
machinery
• Different types/lengths of plasmids
https://www.youtube.com/watch?v=z0PMLofObxk
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Rolling circle replication
Conjugative plasmid
keywordsking.com
learning.uonbi.ac.ke
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Transcription
• RNA synthesis from rNTP on DNA templates by DNAdependent
RNA-polymerase
• Rules of base complementarity
• Essential for life – gene expression
DNA
RNA
Protein
Transcription
Translation
Reverse transcription (retroviruses, …)
Nucleic
acids
Replication
Replication
MB-3-2022 24
Transcription
Messenger RNA (mRNA)
• Transcription of genetic information in structural genes (then translated to amino acids in proteins)
Precursor ribosomal RNA (pre-rRNA)
• Primary transcript for rRNA, subsequently posttranscriptionally edited to rRNA
Precursor transfer RNA (pre-tRNA)
• Primary transcript for tRNA, subsequently posttranscriptionally edited to tRNA
Primary transcripts of regulatory RNAs
Different types of transcripts are synthesized in transcriptional units
MB-3-2022 25
gene X
Prokaryotic genes do not contain introns
gene X
transcript of gene X
AUG
DNA
mRNA
protein X
TRANSCRIPTION
TRANSLATION
Protein
AUG
DNA
mRNA
protein X
TRANSCRIPTION
TRANSLATION
Protein
transcript of gene X
SPLICING
mRNA
gene X
Eukaryotes
Prokaryotes
Introns
Exons
MB-3-2022 26
Prokaryotic genes are polycystronic
gene
A
AUG
DNA
mRNA
one transcript
TRANSCRIPTION
TRANSLATION
Protein
DNA
TRANSCRIPTION
Protein
Eukaryotes
Prokaryotes
Cistron ~ gene
gene
B
gene
C
gene
D
A B C D
A
B
C
D
gene
A
gene
B
gene
C
gene
D
AUG AUG AUG
AUG
TRANSLATION
A
B
C
D
A
B
C
D
AUG
AUG
AUG
mRNA
every gene has
its own transcript
MB-3-2022 27
Transcriptional unit
Transcription occurs in forms of transcriptional units – polycystronic RNA
• Non-operon transcriptional units
• Operons
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Non-operon transcriptional unit
promoter gene A gene B gene C terminator
Last
nucleotide of
terminator
Initation
codon
gene A gene B gene C terminator
AUG AUG AUG
DNA
RNA – primary transcript
RNA
polymerase
TRANSCRIPTION
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Operon
promoter gene A gene B gene C terminator
Last
nucleotide of
terminator
Initiaton
codon
AUG AUG AUG
DNA
RNA – primary transcript
RNA
polymerase
TRANSCRIPTION
• PROMOTER CAN OVERLAP WITH OPERATOR
operator
REPRESSOR
gene A gene B gene C terminator
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Prokaryotic promoter
promoter TATAATTTGACA
-35 -10 +1
gene
-35 element Pribnow box
consensus sequences
• Consensus sequences are „average“ of all promoters
• Promoter similarity ensures affinity to one RNA polymerase
• Differences in consensus sequences modify affinity to RNA polymerase = promoter strength = how often will
transcription be initiated from the promoter – accurate consensus sequences are rare
• Strong bacterial promoter = more similar with consensus
• Weak bacterial promoter = differs more from consensus
• More detailed regulation – substitutions in -35 and -10 elements, distance of -35 and -10 elements, additional
elements in some promoters
(transcription start site = TSS)
downstreamupstream
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Prokaryotic RNA polymerase
• Recognizes promoters of all transcriptional units
• Consists of 5 types of subunits
2x α
• 40 kDa – maintains the stability of complex
1x β
• 155 kDa – key subunit for rNTP binding to enzyme
1x β‘
• 160 kDa – key subunit for promoter binding
1x ω
• 160 kDa – regulation and stability
1x σ
• 85 kDa – σ-factor (sigma factor) – key subunit for promoter binding few
„exchangeable“ variants, for different types of promoters
• Polymerization speed cca 15-20 nt/s – depends on σ-factor
MB-3-2022 32
Transcription progress
Initiation of transcription
Binding of RNA polymerase to promoter – template DNA strand
• Binding of first and second NMPs – rules of complementarity
Elongation
• Begins by creating of first phosphodiester bond
• Connection of nucleoside monophosphates to growing RNA strands
Termination of transcription
• RNA polymerase stalling
• Release of RNA
• Release of RNA polymerase from DNA
MB-3-2022 33
Initiation of transcription
1. Binding of RNA polymerase on -35 element and Pribnow
box (closed transcriptional binary complex)
2. Abolishment of hydrogen bonds in Pribnow box (open
transcriptional binary complex) – transcriptional bubble
3. Transcription of first two nucleotides (open transcriptional
ternary complex)
The β subunit 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 σ subunit. Base −6G is at the σ-β subunit interface. Base +2G
interacts solely with the β subunit (insert).
(Karpen a deHaseth, Biomolecules, 2015)
MB-3-2022 34
Elongation
1. Formation of the first phosphodiester bond
2. Connecting of nucleoside monophosphates to the 3 'end of
the newly synthesized RNA molecule - energy from rNTP
cleavage to rNMP + PP
3. The nucleotides are joined according to the rules of
complementarity of the bases according to the sequence of
the template strand (similar to replication)
4. After the first 10 nucleotides are added, the σ-factor will be
released - the stability of the polymerase and matrix DNA
complex will increase
MB-3-2022 35
Termination of transcription
1. RNA polymerase stalling
2. Release of synthesized RNA
3. Release of RNA polymerase from DNA
Termination of transcription occurs in terminator of transcription:
1. Terminator independent of ρ-factor (rho) - terminator type 1
2. Terminator dependent on ρ-factor (rho) – terminator type 2
MB-3-2022 36
Terminator independent of ρ-factor
• The DNA matrix and therefore the terminator transcript
contains palindromic GC-rich followed by poly-U sequences
• After transcription, synthesized RNA results in the formation
of a hairpin with a loop that slows the RNA polymerase
• The RNA polymerase synthesizes the poly-U region and drops
out of the DNA
• Free RNA polymerase is re-associated with σ-factor
MB-3-2022 37
Rho-dependent transcription termination
• Structure similar to a ρ-factor independent termination
• The DNA matrix and therefore the transcription terminator
contains palindromic GC-rich region
• After transcription, synthesized RNA results in the formation of a
hairpin with a loop that slows the RNA polymerase
• The poly-U sequence is replaced by another - the polymerase
has no release signal
• The newly generated RNA is bound by ρ-factor at the rut site ,
which slides towards the end of the RNA and RNA polymerase
• After contact of the ρ-factor with the RNA polymerase, the
polymerase is released
• Free RNA polymerase is re-associated with σ-factor
MB-3-2022 38
Transcription of structural genes
• The structural genes contain the so-called leader sequence (structural genes only) - before the first structural
gene within the transcription unit - behind the promoter / operator
• The leader sequence (Shine-Delgarn sequence) AGGA (GGU) (RNA) sequence - facilitates binding of RNA to
ribosome to the 3'-end of 16S rRNA
promoter gene A gene B gene C terminatoroperator
Leader
sequence
MB-3-2022 39
Transcription of mRNA - summary
• The primary transcript of the transcription unit containing the
structural genes:
• Contains a leader sequence at the 5 'end
• It contains transcripts of structural genes that are translated into the
polypeptide chain
• At the 3'-end UUUUUUUU = end sequence
• Contains transcriptions of several genes = polycistronic (multigenic)
mRNA
• Posttranscriptionally not modified - does not contain introns = no
splicing
• Life span for several minutes, ribonuclease decomposition in 5'-3'
• At any given time, it accounts for 3% of total RNA in the cell
MB-3-2022 40
Coupling of transcription with translation
• Coupled synthesis
• Ribosomes are already attached to mRNA during
transcription
• On the same mRNA molecule both processes
(transcription + translation) are simultaneous +
degradation of mRNA from the beginning of transcription
• For some transcription units up to 15 transcription
initiations per minute = 15 new mRNA molecules
• For each mRNA up to 30 ribosomes = 30 new
polypeptide chains
• Binding and sliding of ribosomes along mRNA increases
the synthesis rate of RNA polymerase
• Absence of ribozomes, on the other hand, causes slowing
of RNA polymerase, waiting for ribosomes
MB-3-2022 41
Transcription of functional RNAs
• Transcription proceeds from several rRNAs (7 in E.
coli) to form precursor rRNA (pre-rRNA)
• Each transcript contains 16S, 23S and 5S rRNA along
with several tRNAs
• Pre-rRNA is posttranscriptionally methylated and
cleaved by RNase (RNase III) to the respective rRNA
and tRNA
MB-3-2022 42
Translation
• Synthesis of the polypeptide chain (protein, amino acid sequence)
according to the nucleotide sequence in mRNA
DNA
RNA
Protein
Transcription
Translation
Reverse transcription (retroviruses, …)
Nucleic
acids
Replication
Replication
MB-3-2022 43
Genetic code
• the genetic code is a triplet code - one amino acid in the protein is encoded by a
sequence of three nucleotides
• the genetic code is universal - the meaning of individual triplets is almost always
universal and therefore independent of the species of organism
• the genetic code is degenerate - one amino acid can be encoded by several
different triplets (but not vice versa)
Arginine
CGU
CGA
CGC
CGG
AGG
AGA
CGUGGUACGAUUGGAUGU
CGU = Arginine CGU = Arginine CGU = Arginine
Arg Gly Thr Ile Gly Cys
mRNA
Protein
Triplet – Codon x anticodon = complementary
sequence on tRNA carrying aminoacid
MB-3-2022 44
Genetic code
Second nt
First nt U C A G Third nt
U
Phe Ser Tyr Cys U
Phe Ser Tyr Cys C
Leu Ser STOP STOP/Sel A
Leu Ser STOP Trp G
C
Leu Pro His Arg U
Leu Pro His Arg C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
A
Ile Thr Asn Ser U
Ile Thr Asn Ser C
Ile Thr Lys Arg A
Met/START Thr Lys Arg G
G
Val Ala Asp Gly U
Val Ala Asp Gly C
Val Ala Glu Gly A
Val Ala Glu Gly G
MB-3-2022 45
Translation progress
Activation of aminoacids
• Binding of amino acids to tRNA by aminoacyl-tRNA synthetases
Initiation of translation
• A sequence of actions to create the initiation complex - 70S ribosome, mRNA, initiation tRNA - initiation factors
Elongation
• Polypeptide chain extension - elongation factors
Termination of translation
• Termination of synthesis on a senseless codon, release of the polypeptide from the ribosome - termination factors
MB-3-2022 46
Translation conditions in prokaryotes
• mRNA template
• 22 standard aminoacids
• tRNA
• Aminoacyl-tRNA-synthetase
• Ribosomes
• Translation factors
• ATP and GTP
MB-3-2022 47
tRNA• 74 to 95 nucleotides
• MW = 80 000
• 3´- end = CCA
• part of the sequence are unusual bases arising from enzyme
modification after transcription (dihydrouridine, pseudouridine)
• names: tRNA: tRNAAla, tRNALeu
tRNA + AA: Ala ~ tRNAAla, Leu ~ tRNALeu
Dihydrouridine
loop
Pseudouridine
loop
MB-3-2022 48
CCA end of tRNA
• this triplet of nucleotides is present at the 3-end of each tRNA
• on the last A the amino acid is bound during activation
• CCA is added by nucleotidyltransferases using ATP and CTP in post-transcriptional modifications of the
primary transcript
• nucleotidyltransferases are in two classes:
• class I – Archae
• class II - prokaryotes a eukaryotes
• Nucleotidyltransferases recognize immature tRNA (does not have CCA) from mature (CCA-terminated) and
catalyze only immature tRNA
MB-3-2022 49
Aminoacids
MB-3-2022 50
Aminoacyl-tRNA-synthetases
• Enzymes catalysing the covalent binding of the amino acid to the corresponding
tRNA
• M = 40 to 100 kDa
• low homology in the primary structure, only a few conservative sequences
• binding site for amino acid, tRNA and ATP
• tRNA binding site binds related tRNAs
• generally, each amino acid has its aa-tRNA synthetase
• in some bacteria there is one synthetase that binds more amino acids to the
corresponding tRNA
• each tRNA is specific for a single amino acid (not vice versa - each amino acid can
be linked to more than one tRNA)
MB-3-2022 51
Activation of AA - Aminoacyl-tRNA
reaction is catalyzed by aminoacyl-tRNA synthetases
High-energy bond
MB-3-2022 52
Prokaryotic ribosome
70S; 2500 kDa
50S; 1600 kDa
large subunit
30S; 900 kDa
small subunit
Prokaryotic ribosome
34 proteins
23S rRNA
2900 nt
5S rRNA
120 nt
21 proteins
16S rRNA
1540 nt
MB-3-2022 53
Prokaryotic ribosome
• The ribosomal subunits are composed separately after the synthesis of the individual
components
• The subunits associate together with the synthesis of the protein by binding to mRNA near
the 5'-end of the mRNA
• The small subunit provides pairing of anticodon (tRNA) - codon (mRNA)
• The large subunit provides the synthesis of peptide bonds
• The key catalytic activities of the ribosome are assured by rRNA - ribozyme
• Bacterial ribosomes synthesize a binding of approximately 20 amino acids per second
• 4 key binding sites:
• 1x for mRNA
• 3x for tRNA – A (acceptor / aminoacyl), P (peptidyl) a E (exit) site
• Additional binding sites for translational factors (initiation and elongation)
MB-3-2022 54
Nobel Prize in Chemistry
The Nobel Prize in Chemistry 2009 was awarded jointly to Venkatraman Ramakrishnan, Thomas A.
Steitz and Ada E. Yonath "for studies of the structure and function of the ribosome."
<https://www.nobelprize.org/prizes/chemistry/2009/summary/>
Venkatraman Ramakrishnan
Affiliation at the time of the award:
MRC Laboratory of Molecular
Biology, Cambridge, United Kingdom
Thomas A. Steitz
Affiliation at the time of the award:
Yale University, New Haven, CT, USA ,
Howard Hughes Medical Institute
Ada E. Yonath
Affiliation at the time of the award:
Weizmann Institute of Science,
Rehovot, Israel
MB-3-2022 55
Prokaryotic translation
https://www.youtube.com/watch?v=KZBljAM6B1s
Initiation of translation
• Creation of a ternary complex : fMet (N-Formylmethionine) ~ tRNAfMet + GTP + IF2
• Identification of the Shine-Delgarno mRNA sequence associated with the IF3, the 30S ribosomal subunit - the
exact localization of the AUG codon mRNA into the ribosome P site by the complementarity of the ShineDelgarno
sequence and the 16S rRNA region
• Binding of the ternary complex via tRNA-fMet to the ribosome P-site - IF1-containing formation of the preinitiation
complex
• Cleavage of GTP to GDP + Pi, releasing IF1-3, and initiation complex formation with the 50S subunit where fMet ~
tRNA-fMet is at the ribosome P site
Elongation
• Repeated binding of tRNAaa to the ribosome A site with EF-Tu and GTP, creation of a peptide bond between aa at
the P and A site, ribosome translocation (content of A to P, P to E) with EF-G and GTP
Termination of translation
• Recognizing a senseless codon at the A site by RF factor
• Cleavage of the synthesized peptide
• Release of tRNA, mRNA and dissociation of ribosomal subunits
MB-3-2022 56
50S
Prokaryotic translation - initiation
Creation of a ternary complex: fMet ~ tRNAfMet + GTP + IF2
fMet ~ tRNAfMet
IF2
IF2
Binary complexTernary complex
GTP
30S
APE
IF3
Shine-Delgarno
sequence
AUG
30S
APE
IF3
IF2
GTP
16S rRNA
30S
APE
IF3
IF2
GTP
Preinitiation complex
IF1
IF1
30S
APE
GDP
Initiation complex
IF1
+ Pi
Identification of the Shine-Delgarno mRNA sequence associated with the IF3, the 30S ribosomal
subunit - the exact localization of the AUG codon mRNA into the ribosome P site by the
complementarity of the Shine-Delgarno sequence and the 16S rRNA region
Binding of the ternary complex via tRNAfMet to
the ribosome P-site - IF1-containing formation of
the pre-initiation complex
Clearing GTP to GDP + Pi, releasing IF1-3, and
initiating complex formation with the 50S subunit
where fMet ~ tRNAfMet is at the ribosome P site
fMet
50S
MB-3-2022 57
AUG codon and formylmethionin
AUG codon
• It encodes the first amino acid in the peptide
• It also occurs within the mRNA chain
• AUG encodes for methionine, but at the beginning of the
polypeptide is formylmethionine
• There are 2 different methionine tRNAs – tRNAMet a tRNAfMet
• At the beginning of mRNA Met ~ tRNAfMet binds AUG codon which is
formylated to fMet ~ tRNAfMet by IF2
• Inside mRNA Met ~ tRNAMet binds AUG codon via EF-Tu
MB-3-2022 58
Elongation of polypeptide chain
1. aminoacyl-tRNA binds to the A site and the
tRNA used is released from the E site
2. A new peptide bond is created between the
amino acids at the E and at the A site
3. The large subunit of the ribosome moves and
the so-called hybrid sites P / A and E / P are
created
4. The small subunit moves one codon – opens the
A site, tRNAs are shifted from P to E and from A
to P
MB-3-2022 59
Elongation of polypeptide chain
• Binding of aminoacyl-tRNA to site A is mediated
by the elongation factor ET-Tu, which is "driven"
by hydrolysis of GTP to GDP (hydrolysis catalyses
conformational protein change)
• EF-Tu increases the speed and accuracy of
translation - proofreading mechanisms
• EF-G elongation factor catalyses large ribosome
subunits movement - energy from GTP
hydrolysis
MB-3-2022 60
Wobble base pairing
• 22 AA – 64 codons – 40 tRNA
• Some tRNAs bind to multiple codons
• In 1966 F. Crick postulated the wobble hypothesis
• The 1st and 2nd nucleotide (5'-XXo-3 ') are strictly
bound by Watson-Crick pairing to tRNA
• 3rd nucleotide (5'-ooX-3 ') binds alternatively to
non-Watson-Crick pairing
• tRNAs carrying the same AA but recognizing the
different codons are called isoacceptor
MB-3-2022 61
GTPase and GTP
• The process involves GTPase, also a part of the EFTu
factor
• The GTP-binding domain is evolutionarily highly
conserved and occurs from bacteria to higher
eukaryotes
• GTP hydrolysis is associated with a conformational
change involving A2662 from 23S-rRNA
R. M. Voorhees et al., Science 330, 835-838 (2010)
• His84 acts as a base that
removes the proton from H2O
• OH- attacks γ-phosphate in GTP
• GDP is released
MB-3-2022 62
Termination of translation
• the presence of an nonsense codon where AA does
not bind, but the termination factors do
• presence of termination factors RF1 (for UAG and
UAA), RF2 (for UGA and UAA) and RF3 (stimulates the
effect of RF1 and RF2)
• From the carboxyl terminus of the polypeptide chain,
the tRNA is released to stop its elongation
• This frees both the polypeptide chain and the
ribosome, which then breaks down into its subunits
MB-3-2022 63
Regulation of translation speed
A – standard ribosome
movement
B – slowing of ribosome due
to codons for less frequent
AA
C – slowing of the ribosome
due to secondary mRNA
structure
D – slowing of the ribosome
due to positively charged AA
- electrostatic interactions
reduce the speed of
movement in the tunnel
MB-3-2022 64
Mechanism of peptide bond formation
• P-loop and the A-loop are part of 23S-rRNA
• There is no protein in the vicinity of 18A
from the catalytic site
• N3 nitrogen of adenine A2451 is decisive
MB-3-2022 65
Genes are expressed differently
MB-3-2022 66