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3/6/2016
• Protein synthesis
Food Biotechnology-3
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Flow of genetic information is
divided into three steps
Genes and genetic code
Gen: DNA fragment carrying the the genetic information is called as gene. Genes
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are compose of DNA.
• Genetic code is determined by the orders of A,G,T, and C bases.
• Information on DNA is transferred to RNA, and transformad to proteins by the
help of RNAs
Moleküler biyolojinin
santral dogması
• DNA, RNA and protein are called as informational macromolecules, since they
all contain genetic information.
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• Order of amino acids in a polypeptide chain is dtermined by the
order of bases in mRNA.
2) Transcription (copying): Genetic information
contained in DNA is copied in the form of RNA.
This replication process is called as
transcription. RNA molecule that copies the
DNA code is messeger RNA (mRNA).
• Some genes contain codes necessary for
Transfer RNA (tRNA) and ribosomal RNA (rRNA)
These also take part in protein synthesis,
however, they do not contain the codes forr
protein synthesis.
3) Translation: process of protein synthesis using
the information copied on mRNA
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As translation process is started by
mRNA, genetic code is written in
the form of mRNA, not DNA.
• There is a linear relation between the order of aminoacid of a
polypeptide and base order of a gene.
With 4 different nucleotides, a
code of 3 nucleotide could code
maximum 64 codons (43).
• Each 3 bases in mRNA encodes an aminoacid. The nucleotide triplets
in mRNA that encode amino acids are called as codons.
It has been demonstrated thaat
only, 61 codons encode
aminoacids. The rest 3 are stop
codons and do not encode a.a.
20 aminoacids are encoded by codons, so
one amino acid can be encoded by several
codons.
• Each codon encodes a specific amino acid
• Genetic code is translated to proteins. Translation system consists of
ribosomes (protein and rRNA), tRNA and some enzymes
Thus, if amino acid sequence of a protein
molecule is known, it is impossible to
predict the codons.
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On the other hand, If DNA sequence
of a gene is known, amino acid
sequence of the protein can be
known.
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Protein synthesis
PROTEİN SYNTHESIS
• Proteins synthesis is a very complex precess, however it is a
very rapid process. E.g. İn E. coli ribosomes, a polypeptide
chain having 100 amino acids is synthesized in 5 seconds.
• In a living cell, many metabolic reactions are carried out by proteins
• Proteins are one of the most important elements of the living organisms.
E.g:
Blood plasma prroteins
Hormons
Anticores
Enzymes
Chloroplast
Mithocondria
Cell wall proteins
• In a young cell, there are 5.000-50.000 ribosomes, at which
hundreds and thousands of proteins are synthesized
simultanously.
• Protein sysnthesis is controlled by some other molecules (tiem
and amount). So in the protein synthesis many components
take part, not only the translation system components.
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Steps of protein synthesis
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1) Transcription : synthesis of RNA
1)Transcription: The process in which a particular fragment of DNA is copied
into RNA is calle as transcription.
3 key differences between DNA and RNA are:
• RNA contains ribose instead of deoxyribose
• RNA contains uracil instead of thymin
• RNA is single strand
3 types of RNA is produced with transcription.
• mRNA is the reoxy copy of gene on DNA. It carries the information from
DNA to ribosomes.
• Enzymes that attack on DNA do not effect RNA
• Enzymes that attack on RNA do not effect DNA
• Both T and U can bind with A, so change of base do not
effect base pairing.
• tRNA and rRNA are involved in the second step of protein synthesis
(translation).
2) Translation: protein synthesis is realized by using RNAs.
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• Trancription of genetic information from
DNA to RNA is realized by enzyme RNA
polymerase.
• RNA polymerase catalysis the formation
of phosphodiester bonds
• The direction of the
synthesis is from 5’ to 3’,
same as in DNA
synthesis. And the
templete is antiparallel
to the newly synthesized
chain.
• The enzyme requires DNA as templete.
• Substrates of the enzyme are ATP, GTP,
UTP and CTP .
• RNA polymerase adds ribonucletide
triphosphates to the 3’-OH group of the
previously added nucleotide . Energy
(pyrophosphate) generated by the
hydrolysis of NTPs is used for the rowth of
the chain.
• Unlike DNA polymerase,
RNA polymerase does
not require primers.
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RNA polymerases
• Templete of RNA polymerase is double strand DNA molecule.
• Only one strand is trascribed for a gene. However, genes can be any of
the strands. So transcription is realized on both of the strands.
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Bacterial RNA polymerase has the simplest structure and is known in detail.
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RNA polymerase of E.coli has 4 different subunits (polypeptide chain) which are β,
β’,α, σ (sigma).
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Subunits combine to make RNA polymerase haloenzyme which is the active form.
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However, σ factor is not bind tightly, and it can easily remove the haloenzyme
structure. The resulting structure after the removal of σ factor is called as RNA
polymerase core enzyme (α,β,β’).
• These principles are valid for RNA polymerase in all organisms. However,
there differences in eucaryotes and procarotes:
 Procaryotes have one RNA polymerase
 Eucaryotes have 3 RNA polmerases (l, ll, lll) all of which involve in the
trbscription of different genes.
Role of sigma factor is the
recognition of the correct site
where RNA synthesis starts
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Promoters
• Poromoter is 35 bases upstream of the start of trancription sites. This is
called as -35 position. promoter sequence is not transcribed.
• There are two specific sequence (consensus sequences) on the promoter
region which provide the recognition and binding of the enzyme to the
promoter for the initiation of the transcription. These are:
 5’-TTGACA-3’ is in the -35 position
 5’-TATAAT-3’ is in the -10 position (TATA box or Pribnow box)
• For the initiation of RNA synthesis, firstly, RNA polymerase must
recognize the appropriate synthesis sites. These sites are known
as promoter.
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Termination of
Transcription
• -35 consensus
sequences
determined in E.
Coli
• All the sequences
are recognized by
sigma factor
For the safety of protein synthesis,
termination of the transcription is
important as well as the initiation process.
There are several ways for the termination
of transcription:
Hair pin formation:
There are specific base sequences on DNA
that help termination.
• In procaryotes, there are sequence
composed of around 40 bases. First bases
of this sequence is complementer with
last ones, but not the center. So these
bases on RNA make pairs (form double
strand) which has the shape of an hair pin.
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Generally there are Uridins after such
sequences. So, this helps RNA to leave
from DNA.
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Life of Messenger, Ribosomal and Transfer RNA
• Most genes (mRNA) encodes proteins. And, some of them
encodes rRNA and tRNA which are also involved in protein
synthesis.
Rho-ependent
termination
• There are special
proteins for this type of
termination.
• In E.coli a proteins called
as Rho makes the
termination.
• Life-spans of mRNAs in procaryotes are very short (a few
minutes). At the end of this time, they are digested with
ribonucleases.
• tRNA and rRNA are stable due to the their folded structures.
• mRNAs are not folded.
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2) Translation
Ribosomes
• Ribosomes are the sites where triplets in mRNA are translated
to amino acids in a specified order. Thus ribosomes are
considered as translational apparatus.
• Translation is the process by which proteins are synthesized
using the genetic information transcribed on mRNA.
• Translation process can be divided into 5 steps all of which
require various molecules and factors.
a) activation of amino acids
b) initiation of polypeptide synthesis
c) Elongation of polypeptide chain
d) termination of polypeptide synthesis
e) Mpdification on the synthesized chain
• Ribosomes are not selective for any kind of translational
processes. They translate any kind of genetic information that
arrives to the ribosomes.
• tRNA molecules are the adaptors that bring the correct amino
acids with the proper codons in mRNA.
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• Ribosomes are one of the the most
complicated organels in the cell.
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• Eucaryotes have 80S, procaryotes have 70S ribosomes.
• Ribosomes are mainly composed of RNA
and proteins.
PROCARYOTES (70 S ribosome)
EUCARYOTES (80S ribosome)
• There are variety of proteins which make
up 60% of ribosomes.
Large Subunit
(50S)
Small Subunit
(30S)
Large Subunit
(60S)
Small Subunit
(40S)
23S rRNA (2904
nucleotide)
+ 31 protein
16S rRNA (1541
nucleotide)
+ 21 protein
28S rRNA (4718
nucleotide) + 49
protein
18S rRNA (184
nucleotide)
+ 33 protein
• Procaryotes have 52, eucaryotes have
more than 82 different proteins.
• Eventhough, the sizes and number of
ribosomes in eucaryotes and procayotes
are different, their main structures are
similar.
5S rRNA (120
nucleotide)
5.8S rRNA (120
nucleotide)
• Both types have one large and one small
subunits.
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S: Svedberg unit
(10-13 sn
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Transfer RNA
• Each tRNA carries different amino acids to the
ribosomes.
• Sedimentation coefficents are 4 S.
• They are the smallest nucleic acids
in the cells (73-93 nucleotide).
• There is at least one tRNA to carry one amino
acid.
• Some amino acids can be carried with more
than one tRNA (2 or 3).
• 50-70% of the bases make internal
pairings where molecule becomes
double stranded.
• Procaryotes have 60 and eucartotes
have around 100-110 different
tRNA.
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• All tRNAs contain 3 unpaired nucleotides on the acceptor
stems.
• tRNAs contain some spesific nucleotides which
are formed by chemical modification of normal
nucleotides (pseudouridine, inosine,
dehydrouridine, ribothymidine, methyl
guanosine, methyl inosine)
• These unpaired nucleotides are CCA in all tRNAs.
• Amino acid binds to the 3’ OH ribose of A with covalent ester
bond (COOH group of amino acid bind with 3’ OH group of
ribose).
tRNA has cloverleaf structure. It consists of 4 parts:
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•
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Anticodon arm
Acceptor stem
TψC arm
D arm
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a) Activation of amino acids
• Anticodon arm contains triplet bases which are complementer
to the codons in mRNA. These triplets are called as
anticodons.
• Enzyme amynoacyl transferase (amino acyl trna synthetase)
provides matching the amino acids with proper tRNAs.
• Anticodon arm is the most variable part of tRNAs.
• Other parts of tRNA interacts both with ribosomes (mRNA and
proteins) and translation proteins to activate the enzyme
synthetase.
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• Recognition sites on tRNAs
for some amino acids
• Fistly, enzyme activates the amino
acids by reacting them with ATP.
And amino acyl-AMP is formed.
Amnino asit + ATP
aminoaçil-AMP + P-P
• In the second step, activated amino
acid is transferred to the tRNA.
aaminoaçil-AMP + tRNA
Aminoaçil TRNA + AMP
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b) Initiation of Translation
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• tRNA formyl transferase catalyses the addition of formyl group to
methionine.
• The translation always starts with methionine (AUG). It
is formyl methionine in prpcaryotes (fmet).
• The start codon is AUG which encodes methionine.
• This modificatin is realized after the transfer of methionine to tRNA.
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Fmet is encoded by the same codon with the methionine (AUG). Fmet is
used at the beginning.
• AUG is the only codon that encodes the methionine.
• In procaryotes, initiation of protein synthesis is done
on free 30S ribosome subunit.
• During the initiation, 30S ribosome subunit, fmet tRNA,
and initiation proteins IF1, IF2 and IF3 form initiation
complex.
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• Formyl group provides the attachment of methionine to the
specific peptidyl site (P site) on 50 S ribosome subunit.
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Shine Dalgarno sequence
• In addition, it prevent the first a.a (methionine) to be added as an
amino acid of synthesized polypeptide chain.
• Formyl group is removed after the tRNA attaches to the initiation
codon. Thus, the first a.a becomes methionine.
• After the protein synthesis is completed, methionine is usualy
removed from the protein by methionine aminopeptidase.
• It is the sequence which is located around 5-8 bases upstream
of the start codon. It is rich in purine bases.
• 5’-AGGAGGU-3’
• The sequence is not translated.
• It is the ribosimal binding site that assure the initiation of the
translation from the correct codons.
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c) Elongation
• Complementer of this sequence, 3’-UCCUCCA-5’, is in the 16S rRNA and
mRNA and 16S rRNA bind with each other by these sequences.
• So, intiation codon (AUG) finds its correct site. Once, ribosome is attached
to the Shine –Dalgarno site, initiation codon can be found.
• mRNA binds to 30 S subunit of
ribosome.
• tRNAs interact with two sites at
50S subunit
• A-site: acceptor site
• P-site: peptide site (where fmettRNA binds)
• A-site: tTRNA carries the amino
acid attaches (which is the
second codon on mRNA)
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• Formation of peptide bond between two amino acids is catalyzed by the enzyme
peptidyl transferase (which is the component of 23 S region).
• Enzyme breaks the ester bonds of the first amino acid on trna, and binds it with
the amino group of the second a.a to form a peptide bond.
• Factors named as EF-Tu, EF-Ts and EF-G are
required for elongation of the chain.
• EF-Tu provides the transfer and attachment of
the amino acyl tRNA to A-site.
• A GTP molecule is required for this.
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Translocation
• Unloaded tRNA moves to the exit site of the ribosome snd
leaves.
• Process of movement of trna from A-site to P-site is called as
translocation.
• For translocation, EF-G factor and a GTP are required.
• During translocation ribosomes move (not mRNA).
• At each movement process, ribosome moves 3 nucleotides by
which it bind with a new codon at A-site.
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• Tek bir mRNA molekülü, birbirini takip eden çok sayıda ribozom
tarafından polizom denilen bir kompleks oluşturarak translasyona
uğrayabilir.
• Polizomlar translasyonun hızını ve verimliliğini arttırır.
• Her bir ribozomun aktivitesi, komşusu olan diğer ribozomdan
bağımsızdır.
• Böylece bir polizom kompleksindeki her bir ribozom birbirinden
bağımsız tam polipeptitleri yapar.
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c) Sentezin sonlanması (terminasyon)
• Protein sentezinin terminasyonu, ribozom
bir anlamsız (stop) kodona geldiği zaman
gerçekleşir.
• Stop kodonlar : UAA, UAG, UGA
• Hiçbir tRNA stop kodonlarına bağlanmaz.
• Salınma faktörü adında özel bir protein (RF
faktörü) zincir sonlama işaretini tanır ve
polipeptiti uçtaki tRNA’dan keserek oluşan
ürünü salar.
• Daha sonra ribozom alt ünitelere ayrılır.
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