Chapter 4: Basic Genetic Mechanisms

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Chapter 4 Basic Genetic Mechanisms
ZHOU Yong
Department of Biology Xinjiang Medical University
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Teaching Requirements

• 1. Mastering definition of DNA transcription and
  DNA replication process of protein synthesis
  process of DNA replication.
• 2. Comprehending process of DNA transcription.
• 3. Understanding what defines the event of which
  DNA strand is to be transcribed replication
  fork, Okazaki fragment, leading and lagging
  strands.

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Chapter 4 Basic Genetic Mechanisms
? DNA Replication
A Overview of DNA Replication B Mechanism of
DNA Replication
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Section A Overview of DNA Replication
DNA replication is triggered by the expression of
all required proteins, such as DNA polymerase,
DNA primase, and cyclin. 
Gene transcription starts from the promoter,
proceeding along one direction, whereas the DNA
replication starts from the replication origin,
proceeding along both directions.  There is only
one replication origin in the genomic DNA of E.
coli, but the eukaryotic DNA contains many
replication origins in each chromosome.
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There is a major difference between DNA
polymerase and RNA polymerase the RNA
polymerase can synthesize a new strand whereas
the DNA polymerase can only extend an existing
strand.   Therefore, to synthesize a DNA
molecule, a short RNA molecule ( 5 - 12
nucleotides) should be synthesize first by a
specific enzyme.  The initiating RNA molecule is
known as the primer, and the enzyme is called
primase. In addition, DNA replication requires
helicase and single strand binding protein (SSB
protein).  The role of helicase is to unwind the
duplex DNA.  SSB proteins can bind to both
separated strands, preventing them from annealing.
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Schematic drawing of the DNA replication
process.  O1, O2, and O3 are replication origins,
each serving a region called replicon(R1, R2, and
R3).
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In E. coli, movement of the growing fork is about
1000 bp per second.  In eukaryotic DNA, the fork
movement is only about 100 bp per second.  This
is probably due to the association of DNA with
histones, which may hinder the fork movement.  In
humans, replication of the entire genome requires
about 8 hours.  In fruit flies, it takes only 3
- 4 minutes. It takes about 42 minutes to
duplicate the entire genomic DNA. 
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Section B Mechanism of DNA Replication
DNA molecules are synthesized by DNA polymerases
from deoxyribonucleoside triphosphate
(dNTP). Both DNA and RNA polymerases can extend
nucleic acid strands only in the 5' to 3'
direction.  However, the two strands in a DNA
molecule are antiparalle.  Therefore, only one
strand (leading strand) can be synthesized
continuously by the DNA polymerase.  How about
the other strand (lagging strand) ?
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Five types of DNA polymerases in mammalian cells
a, b, g, d, and e. 

• The g subunit is located in the mitochondria,
  responsible for the replication of mtDNA.  Other
  subunits are located in the nucleus.  Their major
  roles are given below
• a synthesis of lagging strand.
• b DNA repair.
• d synthesis of leading strand.
• e DNA repair.

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Though eukaryotic DNA polymerases do not contain
a subunit similar to the E. coli b subunit . 
They use a separate protein called proliferating
cell nuclear antigen (PCNA) to clamp the DNA. 
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2. General Features of DNA Replication
DNA Replication Is Semiconservative
The first definitive evidence supporting a
semiconservative mechanism came from a classic
experiment by M. Meselson and W. F. Stahl.
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Most DNA Replication Is Bidirectional
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DNA Replication Is Discontinuous
DNA polymerases can extend nucleic acid strands
only in the 5' to 3' direction.  However, in the
direction of a growing fork, only one strand is
from 5' to 3'.  This strand (the leading strand)
can be synthesized continuously.  The other
strand (the lagging strand), whose 5' to 3'
direction is opposite to the movement of a
growing fork, should be synthesized
discontinuously. 
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Steps in the synthesis of the lagging strand.
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? Gene Transcription
A Overview of Gene Expression B Overview of
Transcription C Gene's Regulatory Elements D
Transcription Mechanisms in Eukaryotes
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Section A Overview of Gene Expression
An organism may contain many types of somatic
cells, each with distinct shape and function. 
However, they all have the same genome.  The
genes in a genome do not have any effect on
cellular functions until they are "expressed".  
Different types of cells express different sets
of genes, thereby exhibiting various shapes and
functions.
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Section B Overview of Transcription
Transcription is a process in which one DNA
strand is used as template to synthesize a
complementary RNA. 
The DNA strand which serves as the template may
be called "template strand", "minus strand", or
"antisense strand".  The other DNA strand may be
termed "non-template strand", "coding strand",
"plus strand", or "sense strand". 
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• Schematic illustration of transcription. 
• DNA before transcription. 
• During transcription, the DNA should unwind so
  that one of its strand can be used as template to
  synthesize a complementary RNA.

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Growth of a nucleic acid strand is always in the
5' to 3' direction. 
This is true not only for the synthesis of RNA
during transcription, but also for the synthesis
of DNA during replication.  The enzymes, called
polymerases, are used to catalyze the synthesis
of nucleic acid strands. RNA strands are
synthesized by RNA polymerases.  DNA strands are
synthesized by DNA polymerases.
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The entire transcription process involves the
following steps
(i) Binding of polymerases to the initiation
site.  The DNA sequence which signals the
initiation of transcription is called the
promoter.  Prokaryotic polymerases can recognize
the promoter and bind to it directly, but
eukaryotic polymerases have to rely on other
proteins called transcription factors. (ii)
Unwinding (melting) of the DNA double helix. The
enzyme which can unwind the double helix is
called helicase.  Prokaryotic polymerases have
the helicase activity, but eukaryotic polymerases
do not.  Unwinding of eukaryotic DNA is carried
out by a specific transcription factor. (iii)
Synthesis of RNA based on the sequence of the DNA
template strand.  RNA polymerases use nucleoside
triphosphates (NTPs) to construct a RNA
strand. (iv) Termination of synthesis.
 Prokaryotes and eukaryotes use different signals
to terminate transcription.  Note the "stop"
codon in the genetic code is a signal for the end
of peptide synthesis, not the end of
transcription.
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RNA Polymerases
The function of RNA polymerases RNA polymerases
can initiate a new strand but DNA polymerases
cannot.  Therefore, during DNA replication, an
oligonucleotide (called primer) should first be
synthesized by a different enzyme. Strand growth
is always in the 5' to 3' direction. 
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Classes of RNA polymerases E. Coli An E. coli
RNA polymerase is composed of five subunits two
a subunits, and one for each b, b', and s
subunit. The s subunit is also known as the s
factor.  It plays an important role in
recognizing the  transcriptional initiation site,
and also possesses the helicase activity to
unwind the DNA double helix.  Nucleotide
synthesis is carried out by other four subunits,
which together are called the core polymerase. 
The term "holoenzyme" refers to a complete and
fully functional enzyme.  Eukaryotes There are
three classes of eukaryotic RNA polymerases  I,
II and III, each comprising two large subunits
and 12-15 smaller subunits.  However, the
eukaryotic RNA polymerase does not contain any
subunit similar to the E. coli s factor. 
Therefore, in eukaryotes, transcriptional
initiation should be mediated by other proteins.
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Classes of eukaryotic RNA polymerases
RNA polymerase II is involved in the
transcription of all protein genes and most snRNA
genes.  It is undoubtedly the most important
among the three classes of RNA polymerases.  The
other two classes transcribe only RNA genes. 
RNA polymerase I is located in the nucleolus,
transcribing rRNA genes except 5S rRNA.  RNA
polymerase III is located outside the nucleolus,
transcribing 5S rRNA, tRNA, U6 snRNA and some
small RNA genes.
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Section C Gene's Regulatory Elements
Transcriptional regulation is mediated by the
interaction between transcription factors and
their DNA binding sites which are the cis-acting
elements, whereas the sequences encoding
transcription factors are trans-acting
elements.  The cis-acting elements 1. Promoters
2.
Enhancers
3. Silencers
4. Response elements
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1. Promoter The most common promoter element in
eukaryotic protein genes is the TATA box, located
at -35 to -20.  Its consensus sequence, TATAAA,
is quite similar to the -10 region of the Sigma
70 recognition site.  Another promoter element is
called the initiator (Inr).  It has the consensus
sequence PyPyAN(T/A)PyPy.  TATA box and
initiator are the core promoter elements.  There
are other elements often located within 200 bp of
the transcriptional start site, such as CAAT box
and GC box which may be referred to as
promoter-proximal elements.
Eukaryotic promoter elements
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2.Enhancers Human b globin gene cluster The
human b globin gene cluster is controlled by an
enhancer region comprising HS1 to HS4, which
contain the binding sites of GATA-1, NF-E2, AP-1
and other transcriptional activators.  This
region is known as the locus control region
(LCR), which regulates the expression of all five
genes (e, Gg, Ag, d and b), even though the
distance between HS4 and the b gene is as far as
60 kb.   In embryonic DNA, the e gene is
preferentially expressed.  In fetal DNA, Gg and
Ag are much more strongly expressed than the
other genes.  In adult DNA, expression is
switched to mainly the b gene, while the d gene
is weakly expressed.
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Section D Transcription Mechanisms in Eukaryotes
general transcription factors
In eukaryotes, these two functions of recognize
the promoter and unwind the DNA double helix are
carried out by a set of proteins called general
transcription factors.  The RNA Pol II is
associated with six general transcription
factors, designated as TFIIA, TFIIB, TFIID,
TFIIE, TFIIF and TFIIH.
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TFIID consists of TBP (TATA-box binding protein)
and TAFs (TBP associated factors).  The role of
TBP is to bind the core promoter.  TAFs may
assist TBP in this process.  In human cells, TAFs
are formed by 12 subunits.  One of them, TAF250,
has the histone acetyltransferase (HAT) activity.
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Assembly of the pre-initiation complex (PIC). 
TBP first binds to the promoter and then
recruits TFIIB to join TFIID (and TFIIA if
present).  Before entering PIC, RNA Pol II and
TFIIF are bound together, which are recruited by
TFIIB.  Finally, RNA Pol II recruits TFIIE, which
further recruits TFIIH to complete the PIC
assembly.
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Elongation After PIC is assembled at the
promoter, TFIIH can use its helicase activity to
unwind DNA.  This requires energy released from
ATP hydrolysis.  The DNA melting starts from
about -10 bp.  Then, RNA Pol II uses nucleoside
triphosphates (NTPs) to synthesize a RNA
transcript.  During RNA elongation, TFIIF remains
attached to the RNA polymerase, but all of the
other transcription factors have dissociated from
PIC. The carboxyl-terminal domain (CTD) of the
largest subunit of RNA Pol II is critical for
elongation.  In the initiation phase, CTD is
unphosphorylated, but during elongation it has to
be phosphorylated.  This domain contains many
proline, serine and threonine residues.
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Termination Eukaryotic protein genes contain a
poly-A signal located downstream of the last
exon.  This signal is used to add a series of
adenylate residues during RNA processing. 
Transcription often terminates at 0.5 - 2 kb
downstream of the poly-A signal, but the
mechanism is unclear.
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Protein Synthesis
a). Overview of translation b). Ribosome c).
Transfer RNA d). Messenger RNA and The genetic
code e). Protein synthesis
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Overview of translation

• last step in the flow of genetic information.
• The synthesis of every protein molecule in a cell
  is directed by a mRNA originally trans-cripted
  from DNA. Polypeptide synthesis includes two
  kinds of pro-cesses
• (1) the information transfer by which the RNA
  nucleo-tide sequence determines the amino acid
  sequence.
• (2) the chemical processes by which the amino
  acids are linked together.
• The complete series of events constitutes
  translation.

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Overview of translation

• requirements for protein synthesis
• mRNA
• ribosomes
• initiation factors
• elongation and termination factors
• GTP
• aminoacyl tRNAs
• amino acids
• aminoacyl tRNA synthetases
• ATP

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Ribosomes
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Ribosome structure
P
P
P
P
Large subunit
P
P
P
A
P
P-site peptidyl tRNA site
A-site aminoacyl tRNA site
mRNA
5
Small subunit
Ribosome with bound tRNAs and mRNA
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Ribosomes
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• Ribosomes
• prokaryotic ribosome
• eukaryotic ribosome

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• Polysomes
• direction of translation is 5 to 3 along the
  mRNA
• direction of protein synthesis is N terminus to
  C terminus

nascent polypeptide
large ribosomal subunit
N
N
UGA
5
AUG
polysome
small ribosomal subunit
subunits dissociate
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Polysomes
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Transfer RNA

• Transfer RNA molecule act as adaptor that
  translate nucleotide sequence into protein
  sequence.
• All tRNAs have a common cloverleaf structure. It
  includes
• acceptor stem
• CCA-3 terminus to which amino acid is coupled
• carries amino acid on terminal adenosine
• anticodon stem and anticodon loop
• D-arm(D-stem and D-loop)
• T-arm(T?C-arm)
• Variable arm

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tRNA
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Modified nucleosides in tRNA
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tRNA
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• Amino acid activation and
• aminoacyl tRNA synthetases
• aminoacyl tRNA synthetases are the enzymes that
  charge the tRNAs
• Specific aminoacyl tRNA synthetases couple each
  amino acid to its appropriate tRNA molecule and
  create an aminoacyl-tRNA.
• one aminoacyl tRNA synthetase for each amino
  acid
• can be several different isoacceptor tRNAs for
  each amino acid
• all acceptor tRNAs for an amino acid use the
  same synthetase.

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amimoacyl-tRNA
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Amino acid activation and aminoacyl tRNA
synthetases

• each aminoacyl tRNA synthetase binds
• amino acid
• ATP
• acceptor tRNAs
• Amino acid are added to the carboxyl-terminal end
  of a growing ploypeptide chain.

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Peptidyl-tRNA
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amino acid
uncharged tRNA
3
ATP
adenylated (activated) amino acid
PPi
AMP
Amino acid activation and tRNA charging
aminoacyl (charged) tRNA
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Aminoacyl tRNA synthetases
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Messenger RNA (mRNA)
initiation codon
Cap
5 untranslated region
5
AUG
m7Gppp
translated (coding) region
UGA
termination codon
3 untranslated region
(AAAA)n
3
AAUAAA
poly(A) tail
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The genetic code

• consists of 64 triplet codons (A, G, C, U) 43
  64
• all codons are used in protein synthesis
• 20 amino acids
• 3 termination (stop) codons UAA, UAG, UGA
• AUG (methionine) is the start codon (also used
  internally)
• multiple codons for a single amino acid
  degeneracy

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The Genetic Code
UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG
GUU GUC GUA GUG
UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG
GCU GCC GCA GCG
UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG
GAU GAC GAA GAG
UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG
GGU GGC GGA GGG
Phe Leu Leu Val
Ser Pro Thr Ala
Tyr Stop His Gln Asn Lys Asp Glu
Cys Arg Ser Arg Gly
Stop Trp
Ile Met
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Codon-anticodon interactions

• codon-anticodon base-pairing is antiparallel
• the third position in the codon is frequently
  degenerate
• one tRNA can interact with more than one codon
  (therefore 50 tRNAs)
• wobble rules
• C with G or I (inosine)
• A with U or I
• G with C or U
• U with A, G, or I
• I with C, U, or A

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Codon-anticodon interactions
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Codon-anticodon interactions
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Codon-anticodon interactions
3
5
tRNAmet
U A C
A U G
mRNA
5
3
3
5
tRNAleu

• one tRNAleu can read two
• of the leucine codons

wobble base
G A U
C U A G
mRNA
5
3
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wobble rules
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Wobble Interactions
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• Reading frame
• reading frame is determined by the AUG initiation
  codon
• every subsequent triplet is read as a codon until
  reaching a stop codon

• ...AGAGCGGA.AUG.GCA.GAG.UGG.CUA.AGC.AUG.UCG.UGA.UC
  GAAUAAA...
• MET.ALA.GLU.TRP.LEU.SER.MET.SER
• a frameshift mutation
• ...AGAGCGGA.AUG.GCA.GA UGG.CUA.AGC.AUG.UCG.UGA.UCG
  AAUAAA...
• the new reading frame results in the wrong amino
  acid sequence and
• the formation of a truncated protein
• ...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUA
  AA...
• MET.ALA.ASP.GLY

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The process of protein synthesis

• The actual mechanism of protein synthesis can be
  divided into three stages
• Initiation - the assembly of a ribosome on an
  mRNA molecule
• Elongation - repeated cycles of amino acid
  delivery, peptide bondformation and movement
  along the mRNA (translocation)
• Termination - the release of the new protein
  chain.

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Initiation of protein synthesis In prokaryotes

• In prokaryotes, initiation requires
• 1.The large and small ribosome subunits
• 2.The mRNA molecule
• 3.The initiator tRNA
• 4.Three initiation factors (IFs)
• IF-1 and IF-3 bind to the 30S subunit and
  prevent the large subunit binding.
• IF-2 can then bind and will help the initiator
  tRNA to bind later.
• 5.GTP

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The initiator tRNA
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2
2 3
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Initiation of protein synthesis In prokaryotes

• 1.Binding of IF-3 to the 30S subunit, which
  prevents reassociation between the ribosomal
  subunits.
• 2.Binding of IF-1 and IF-2, alongside IF-3.
• 3.Binding of mRNA and fMet-tRNA to form the 30S
  initiation complex. IF-2 sponsors fMet-tRNA
  binding, and IF-3 sponsors mRNA binding.
• 4.Binding of the 50S subunit, with loss of IF-1
  and IF-3.
• 5.Dissociation of IF-2 from the complex, with
  simultaneous hydrolysis of GTP. The product is
  the 70S initiation complex.

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Initiation of protein synthesis in eukaryotes

• Several features distinguish eukaryotic
  translation initiation from prokaryotic.
• First, eukaryotic initiation begins with
  methionine, not N-formyl-methionine.
• Second , eukaryotic mRNAs have caps at their
  5'-ends, which direct initiation factors to bind
  and begin searching for an initiation codon.

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Initiation of protein synthesis in eukaryotes
mRNA binding
M
Initiator tRNA bound to the small ribosomal
subunit with the eukaryotic initiation factor-2
(eIF2)
eIF2
40S subunit
The small subunit finds the 5 cap and scans down
the mRNA to the first AUG codon
mRNA
5 cap
AUG
70
60S subunit

• the initiation codon is recognized
• eIF2 dissociates from the complex
• the large ribosomal subunit binds

eIF2
M
mRNA
5
AUG
40S subunit
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Elongation of protein synthesis

• Elongation takes place in three steps
• (1) Aminoacyl-tRNA delivery.EF-Tu, withGTP, binds
  an aminoacyl-tRNA to the ribosomal A site.
• (2)Peptide bond formation.Peptidyl transferase
  forms a peptide bond between the peptide in the P
  site and the newly arrived aminoacyl-tRNA in the
  A site. This lengthens the peptide by one amino
  acid and shifts it to the A site.
• (3)Translocation. EF-G, with GTP, translocates
  the growing peptidyl-tRNA, with its mRNA codon,
  to the P site.

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A
M
AUG
GCC
mRNA
5
M
A

• aminoacyl tRNA binds the A-site
• first peptide bond is formed

mRNA
5
AUG
GCC
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• Peptide bond formation
• peptide bond formation is
• catalyzed by peptidyl transferase
• peptidyl transferase is contained within
• a sequence of 23S rRNA in the
• prokaryotic large ribosomal subunit
• therefore, it is probably within
• the 28S rRNA in eukaryotes
• the energy for peptide bond formation
• comes from the ATP used in tRNA charging
• peptide bond formation results in a shift
• of the nascent peptide from the P-site
• to the A-site

P-site
A-site
N
NH2 CH3-S-CH2-CH2-CH
OC O tRNA
NH2 CH3-CH OC O tRNA
NH2 CH3-S-CH2-CH2-CH
OC
OH tRNA
NH CH3-CH OC O tRNA
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Large ribosomal subunit
23S RNA (orange and white) makes up the core of
the subunit
Protein (purple) lies on the surface

• Structure shows only RNA
• in the active site
• Adenine 2451 carries out
• acid-base catalysis

Cech (2000) Science 289878-879 Ban et al. (2000)
Science 289905-920 Nissen et al. (2000) Science
289920-930
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Translocation
P

• following peptide bond formation
• the uncharged tRNA dissociates
• from the P-site

P
P
P
P

• the ribosome shifts one codon along
• the mRNA, moving peptidyl tRNA
• from the A-site to the P-site this
• translocation requires the
• elongation factor EF2

UCA
GCA GGG UAG
EF1
EF2

• the next aminoacyl tRNA then
• binds within the A-site this tRNA
• binding requires the elongation
• factor EF1

P
P
P
P
P

• energy for elongation is provided by
• the hydrolysis of two GTPs
• one for translocation
• one for aminoacyl tRNA binding

UCA
GCA GGG UAG
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Termination of protein synthesis

• 1.A stop codon is encountered, the tRNA holding
  the polypeptide remains in the P site
• 2.A release factor (RF) binds with the ribosome.
• 3.GTP hydrolysis provides the energy to cleave
  the polypeptide from the tRNA.
• 4.Eject the release factor and dissociate the
  ribosome from the mRNA.,subunits are recycled to
  initiate translation of another mRNA.

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Termination of protein synthesis

• Eukaryotes have only one release factor that
  recognizes all three stop codons UAA,UAG, and
  UGA.
• In prokargotes, the release factor RF-1
  recognizes the stop codons UAA and UAG, release
  factor RF-2 recognizes UAA and UGA.

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Termination
RF
P
P

• when translation reaches the stop
• codon, a release factor (RF) binds
• within the A-site, recognizing the
• stop codon

P
P
P
UCA
GCA GGG UAG
P
P
P
P
P
P
P
P

• release factor catalyzes the hydrolysis
• of the completed polypeptide from
• the peptidyl tRNA, and the entire
• complex dissociates

UCA
GCA GGG UAG
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Inhibitors of protein synthesis
Inhibitor Process Affected Site of Action
Kasugamycin initiator tRNA binding 30S subunit
Streptomycin initiation, elongation 30S subunit
Tetracycline aminoacyl tRNA binding A-site
Erythromycin peptidyl transferase 50S subunit
Lincomycin peptidyl transferase 50S subunit
Clindamycin peptidyl transferase 50S subunit
Chloramphenicol peptidyl transferase 50S subunit
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REVIEW QUESTIONS

• 1. How different sizes of rRNA combine with
  different types of protein to make eukaryotic
  ribosome?
• 2. Compare the process of DNA replication and RNA
  transcription.
• 3. Describe the process of protein synthesis.