B.Sc.

Biotechnology Molecular Biology

Translation in prokaryotes

Praveen Deepak
Assistant Professor of Zoology
Swami Sahjanand College
Jehanabad
 Introduction

 It is the process of synthesis of proteins from messenger RNA transcripts (mRNA)

after the process of transcription of DNA to RNA.
 It takes place in the cytoplasm by specialized organelle known as ribosomes.
 There are no endoplasmic reticulum in the prokaryotes and ribosomes are
suspended in the cytoplasm, whereas endoplasmic reticulum are present in
eukaryotes which harbors ribosomes – translation takes place on rough
endoplasmic reticulum (RER) in eukaryotes, whereas translation occurs freely in
cytoplasm in the prokaryotes.
 The codons on the mRNA are translated into amino acid sequence which leads to
the synthesis of protein.
 Translation requires a variety of cellular components, such as proteins, RNAs and
different small molecules.
 It has also three main steps:
o Initiation – Formation of mRNA-ribosome complex
o Elongation – Formation of polypeptide chain complimentary to the mRNA
o Termination – Termination of polypeptide chain

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 The “Central Dogma”

 Flow of genetic information in a cell

– How do we move information from DNA to proteins?

Francis Crick
1958

Source: AP Biology

“It states that genetic information is transmitted form DNA to RNA to protein and
this information cannot be transferred back from protein to either protein or
nucleic acid”.

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 The “Central Dogma”

Inheritance of metabolic disease

– It suggests that genes code for enzymes.
– Each disease, i.e., phenotype, is caused by non-functional gene
product, which may be
• lack of an enzyme
• Tay Sachs disease - hexosaminidase A, causes a fatty substance to collect .
• Phenylketonuria (PKA) - absence or deficiency of an enzyme called phenylalanine
hydroxylase (PAH), responsible for processing the amino acid phenylalanine.
• Albinism - absence or defect of tyrosinase, a copper-containing enzyme involved in the
production of melanin.
Am I just the
Proteome - between 80,000 and 400,000 proteins. sum of my proteins?

Source: AP Biology

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 Proteins

 Proteins are the “workhorse” molecule found in organisms, which determine

phenotype of an organism, i.e. what we look like.
 Proteins are made of polypeptide chains that have primary, secondary, tertiary and
quaternary structure.
 The polypeptide chains are made of 20 amino acids - an average polypeptide chain is
400 amino acid long (can be shorter than this value).
 The part of the DNA that codes for a particular polypeptide chain is called as a gene.

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Uses of Proteins

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Work of Beadle and Tatum

George Beadle and Edward Tatum

established the link between genes and
enzymes in studying bread mold,
Neurospora crassa.

Conclusion
• One gene produces one enzyme.
• Later it was modified
• One gene produces one protein.
• One gene produces one polypeptide
chain.

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 Overview of Protein Synthesis

In prokaryotes In eukaryotes

 DNA is transcribed to mRNA in the cytoplasm in prokaryotes as it has no nucleus,

and in the nucleus in eukaryotes.
 With mRNA and help of other RNA as well as protein molecules, mRNA is translated
into specific proteins in the cytoplasm
 Therefore, RNA is the intermediate between the DNA code and the actual synthesis
of a protein

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 RNAs in the Protein Synthesis

RNA

 ribose sugar
 N-bases
 uracil instead of thymine

 U : A

 C : G

 single stranded
 lots of RNAs
 mRNA, tRNA, rRNA, siRNA…

transcription
DNA RNA
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 RNAs in the Protein Synthesis
Types of RNA in protein synthesis

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 RNAs in the Protein Synthesis
3 types of RNA help in protein synthesis
Messenger RNA (mRNA)

 It carries code from DNA to ribosomes. It actually acts as a transporter of

genetic information to protein synthesis machinery.
 It is specific for a particular protein.

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 RNAs in the Protein Synthesis
3 types of RNA help in protein synthesis
RIBOSOMAL RNA (rRNA)

 Made in nucleolus in eukaryotes, while

it is made in the cytoplasm in prokaryotes.
 It has 2 subunits (large & small)
 It combines with proteins to form ribosomes
 Prokaryotic ribosomes are of different size
than eukaryotic ribosomes (30S and 50S
subunits in prokaryotes and 40S and 60S Ribosomal subunits – RNA in orange
subunits in eukaryotes). and yellow and proteins in blue.
 Medically significant – some antibiotics Source: https://pdb101.rcsb.org/motm/10
target bacterial ribosomes in the host

Endosymbiotic theory: It states that some of the organelles in eukaryotic

cells were once prokaryotic microbes.

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 RNAs in the Protein Synthesis
3 types of RNA help in protein synthesis
Transfer RNA (tRNA)

 Anticodon sequence are present.

 Anticodon sequence matches

codon on mRNA to add correct
amino acid during protein
synthesis.

 Aminoacyl-tRNA synthetase
enzyme attaches a specific amino
acid using energy from ATP. http://www-math.mit.edu/~lippert/18.417/lectures/01_Intro/

What is the anticodon for the codon AUG?

UAC
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Protein Synthesis in Prokaryotes and Eukaryotes

Difference between Protein Synthesis in Prokaryotes and Eukaryotes

 Prokaryotes do not have introns like eukaryotes.

 There is no or very little RNA processing in
prokaryotes.
 Transcription and translation can be simultaneous in prokaryotes.

Protein synthesis requires genetic information as genetic code contained in the DNA.

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 Genetic Code

Amino acids are coded by triplets

of DNA nucleotides, called codons.

There are 64 codons.

61 code for amino acids, 3 code

for STOP, and one codes for
START and also for Methionine.

Marshall Nirenberg and Heinrich Matthaei determined the first codon for an amino acid, which
was UUU that coded for the amino acid phenylalanine, by creating mRNA entirely of uracil.

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Genetic Code

 The code has redundancy

(GGU, GGC, GGA, and
GGG); all code for the amino
acid glycine.

 Each codon only codes for

one amino acid.

 The code is a universal code

meaning almost all cells use
the same code. A eukaryotic
gene can be expressed in a
prokaryotic cell.

 Code for ALL life (common

origin for all life..

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Translation of Codons into a Polypeptide

The gene designates that the following peptide chain be made with the
amino acids in this particular order.

 Start codon  Stop codon

 AUG  UGA, UAA, UAG

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Why is genetic code triplate?

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How are the codons matched to amino acid?

Charging of tRNA by an enzyme called aminoacyl tRNA synthetase also called

tRNA-ligase (one aminoacyl tRNA synthetase is present for each standard amino
acid, i.e., 20 aminoacyl tRNA synthetase are present in most of the cell).

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 Charging of tRNA

Charging of tRNA is accomplished by an enzyme aminoacyl tRNA synthatse

 Aminoacyl tRNA synthetase

 enzyme which bonds amino acid to tRNA

 bond requires energy

 ATP  AMP

 bond is unstable, so it can release amino acid at ribosome easily

Trp C=O Trp C=O Trp

OH H2O
OH O
O
activating
enzyme

tRNATrp AC C
anticodon UGG mRNA
tryptophan attached
to tRNATrp tRNATrp binds to UGG
condon of mRNA
Ester bond between amino acid and tRNA
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How are the codons matched to amino acid?

 Each codon represents a particular amino acid, and each codon is

recognized by a specific tRNA.
 Only codon specific anticodon containing tRNA interacts with codon,
otherwise tRNA of wrong anticodon cannot interact with codon site.

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 Ribosomes

 Ribosomes are a complex

of ribosomal RNA (rRNA) and
proteins.
 It is composed of two sub-
units of unequal size;
 Large subunit
 small subunit
 It facilitates coupling of tRNA
anticodon to mRNA
 It has many sites meant for
specialized function, which are:
→ A site (aminoacyl-tRNA site)
 It holds tRNA carrying next amino acid to be added to the polypeptide
chain.
→ P site (peptidyl-tRNA site)
 It holds tRNA carrying growing polypeptide chain.
→ E site (exit site)
 It ensures empty tRNA leaves ribosomes from exit site

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 Protein Synthesis

Three steps

 Initiation
 It brings together mRNA, ribosomal subunits and initiator tRNA.
 Elogation
 In this phase, adding of amino acids take place based on codon
sequence.
 Termination
 Here codon ends and translation stops

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 Initiation of Translation

At Shine-
Dalgarno
sequence (also
known as the
ribosomal
binding site
AGGAGG), small
subunit of
ribosome
attaches.

In prokaryotes, protein synthesis begins with the formation of an initiator complex

which involves the small 30S ribosome, the mRNA template, 3 initiation factors that
helps the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as
a energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-
tRNAfMet)

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 Elongation of Polypeptide Chain
 Polyribosomes (Polysomes)
 Numerous ribosomes translate same mRNA at same time

 3-D folding (1’, 2’, 3’ structure)

 Chaparonins: provide essential kinetic assistance to protein folding by binding non-

native proteins.

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 Elongation of Polypeptide Chain

Prokaryotes

 Elongation starts with the occupation of metthionyl-tRNA to the ribosome P – site

after recognizing a start codon on mRNA bound to small ribosomal subunit.
 The large subunit then joins the small subunit, and a second tRNA is recruited.
 As the mRNA moves relative to the ribosome, successive tRNAs move through
the ribosome and the polypeptide chain is formed.
 In this way, elongation proceeds with charged tRNAs sequentially entering and
leaving the ribosome as each new amino acid is added to the polypeptide chain.

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 Elongation of Polypeptide Chain

Souii et. al. International Journal of Molecular Sciences 2013; 14(3):4525-44 Prokaryotes

 This movement of a tRNA from A to P to E site is induced by conformational

changes that advance the ribosome by 3 bases in 3′ direction.
 The energy for each step, such as binding of a new aminoacyl-tRNA to the A site
and for its translocation to the P site after formation of the peptide bond along the
ribosome is donated by elongation factors that hydrolyze GTP.
 Peptide bonds form between the amino group of the amino acid attached to the A-
site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA by
an enzyme peptidyl transferase ntegrated into large subunit..

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 Termination of Translation

Step 1 – After the amino acids needed to make the protein required are
properly bonded to each other a release factor binds to the complex when
a stop codon enters the A site.
Step 2 – The release factor then disconnects the polypeptide from the tRNA in the
P site.
Step 3 – The remaining components mRNA, ribosomal subunit, and the protein are
released and the process of translation ends.

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Overview of Translation

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 Overview of Translation

Comparison of translation in Prokaryotes and Eukaryotes (& Archea)

Ribosomes 70S 80S
 30S (small subunit) with  40S (small subunit) with
16S rRNA subunit 18S rRNA subunit
 50S (large subunit) with  60S (large subunit) with
5S and 23S rRNA 5S, 5.8S and 28S rRNA
subunits subunits
Amino acid carried by fMet Met
initiator tRNA
Shine-Dalgarno sequence Present Absent
in mRNA
Simultaneous transcription Yes No
and translation

In prokaryotes, Met-tRNA is modified by the addition of a formyl group and it

is fMet-tRNA in prokaryotes.

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 Brainteaser

Do the same DNA sequences lead to the same proteins in

all organisms?

o No, due to differential mRNA splicing identical DNA sequences may

lead to non-identical proteins.

o There are about 150 known cases of deviations from the universal
genetic code with 1 or 2 codons being re-assigned to different amino
acids; typically in small (organelle) genomes, but occasionally in
nuclear genomes as well.

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 Further reading

 Willey J., Sherwood L., Woolverton C.J. 2017. Prescott’s Microbiology 10 th Edition,
McGraw Hill Publication, New York, USA

 Krebs J.E., Goldstein E.S., Kilpatrick S.T. 2017. Lewin’s Genes XII. Jones and Bartlett
Publishers, Inc., Burlington, MA, USA

 Snyder L.R., Peters J.E., Henkin T.M., Champness W. 2013. Molecular Genetics of
Bacteria, 2nd ed., ASM Press, Washington DC, USA, 2003.

 Graumann P.L. Chromosome architecture and segregation in prokaryotic cells. Microbial

Physiology 24(5-6).

 Griswold A. 2008. Genome Packaging in Prokaryotes: the Circular Chromosome of E.

coli. Nature Education 1(1):57.

 Kuzminov A. 2014. The precarious prokaryotic chromosome. Journal of Bacteriology

196(10):1793-1806.

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