Role of DNA and RNA in Protein Synthesis

Sardar Vallabhbhai Patel University Of
Agriculture & Technology
Meerut-250110(U.P.)
Master’s Seminar
Course Code:GP-591
Topic :Role of DNA and RNA in Protein Synthesis
Submitted to Submitted by
Dr.S.A. Kerkhi, Charupriya Chauhan
Dr.Pooran Chand, M.Sc.(Ag),GPB
Dr.S.K.Singh, Id.No: PG/A-3392/15
Dr. Mukesh Kumar

Contents
 Role of DNA and RNA in protein synthesis
-Protein synthesis
-Protein synthesis in Eukaryotes and
Prokaryotes
-Central Dogma of molecular biology
 Transcription
-Transcription in Prokaryotes
-Transcription in Eukaryotes
 mRNA processing
 Translation
-Messenger RNA
-Genetic code
-Ribosome and ribosomal RNA
-Transfer RNA
-Formation of aminoacyl tRNA
 Translation process
-Initiation
-Elongation
-Termination
 Conclusion
 References

Role of DNA & RNA in Protein Synthesis
 DNA, is a double stranded nucleic acid consisting of deoxyribose sugar and carries the
genetic instructions used in the development and functioning of all known living
organisms.
 RNA is a single stranded molecule consisting of ribose sugar and it is transcribed
(synthesized) from DNA by enzymes called RNA polymerases. RNA acts as a messenger
between DNA and the protein synthesis complexes known as ribosomes.
 Proteins are one of the vital biomolecules of life. These compounds perform a variety of
essential processes to sustain an organism's survival, which include clotting of blood,
transporting oxygen, contracting muscles and catalyzing chemical reactions. The
building blocks of proteins are called amino acids.

 A typical α-amino acid consists of an amino group (-NH2), carboxyl group (-COOH)
and an R group.
 Amino acids in a protein chain are linked by a peptide bond. Usually, proteins have an
N-terminal end carrying a free amino group and C-terminus with -COOH.

Protein Synthesis
5
The synthesis of proteins starts with transcribing the
instructions in DNA into mRNA.
The mRNA is then carried out of the cell's nucleus into the
cytoplasm, specifically into structures called ribosomes.
Protein production occurs in ribosomes containing rRNA.
The tRNA transports the amino acids to the ribosomes.
The code sequence in mRNA is then translated and
specific proteins are synthesised by stringing amino acids
together.
The production or synthesis of polypeptide chains
(proteins) includes two phases: Transcription &
Translation
DNA
mRNA
tRNA (ribosomes)
Protein
Pathway to Making a
Protein

Protein Synthesis in Eukaryotes & Procaryotes
In eukaryotes, mRNA is synthesized in
the nucleus from pre-messenger RNA
(pre mRNA) molecules, and then
shipped to the cytoplasm, where
translation occurs
In prokaryotes, transcription and
translation occur in the same cellular
compartment — the cytosol. Ribosomes
are the site of translation

Central Dogma
Proposed by Crick in 1958.
Original proposal: Direction of information flow : DNA RNA Protein
But not in the reverse
Current status: General information flow: DNA RNA Protein.
In Case of some viruses: RNA RNA & RNA DNA
RNA
Replication
Reverse Transcription

Transcription
 Transcription is the process through which a DNA sequence is enzymatically copied by
an RNA polymerase to produce a complementary RNA(mRNA). In other words, it is the
transfer of genetic information from DNA into RNA.
 During transcription, only one strand of a DNA molecule is transcribed; this strands is
called antisense strand or template strand and the RNA so produced is termed as sense
RNA.
 The other strand of the DNA duplex is know as coding strand or sense strand.
 As in DNA replication, transcription proceeds in the 5' → 3' direction (i.e. the old
polymer is read in the 3' → 5' direction and the new, complementary fragments are
generated in the 5' → 3' direction).
 Transcription, is catalyzed by DNA-directed RNA polymerase, or simply RNA
polymerase.

Direction of transcription
The DNA splits into two strands:
•Template strand: it is used to synthesize RNA.
•Coding Strand (Informational strand): it is not used to synthesize RNA.
•Transcription proceeds from the 3’ end to the 5’ end of the template..
Process of Transcription

Transcription Unit
A transcription unit is that stretch or sequence of DNA that is transcribed into a
single RNA molecule. A typical transcription unit has :
 a promoter at its beginning (at the 3ꞌ- end of its antisense strand),
 a startpoint,
 a coding region and
 a terminator sequence at its end.
Coding, Plus (+), Sense strand
Template, Minus (-), Antisense strand
Promoter
5'
3'
3'
5'
Double stranded DNA
Transcription in Prokaryotes

 RNA polymerase binds to the promoter, and transcription begins at the startpoint it
progresses through the coding sequence and ends around the terminator site.
 In case of eukaryotes, each gene is a distinct transcription unit. But in case of
prokaryotes each genes encoding the enzymes for single biosynthetic pathway are
usually clustered together into a single regulatory unit called operon , each operon
functions as a single transcription unit.
 The sequences located on the left of startpoint are called upstream sequences, and the
base positions are denoted sequentially as -1, -2, -3, etc.
 But the sequence located to the right of startpoint are termed as downstream sequences,
and the bases are sequentially designated as +1, +2, +3 etc.
 The RNA molecules obtained directly from transcription are called primary transcripts,
they contain all the sequences, beginning from the startpoint to the terminator site,
present in the antisense strand.
 Primary transcripts are unstable, they are either modified, e.g. eukaryotic mRNAs, all
rRNAs and tRNA , or are degraded (prokaryotic mRNAs) rapidly.

Promoter is that sequence of a transcription unit where RNA polymerase binds and
initiates transcription. E. coli promoters have the following 4 consensus sequence :
1. Start point.
2. -35 sequence
3. -10 sequence
4. The distance between -10 and -35 sequence
A consensus sequence is a base sequence that is present in all the promoters (or other
DNA sequences having the same function) but with some variation. In contrast, a
conserved sequences is present, without any change, in all DNA sequence having the
same function.
Optimal Prokaryotic Promoter

Startpoint
Startpoint is the site within a promoter from which transcription begins. It is
typically a single base within the promoter, in >90% of the cases, startpoint is a
purine in the antisense strand. Often startpoint is the A residue within the triplet
CAT.
The -10 Sequence
The midpoint of -10 sequence is, on an average, 10 bp on the upstream (left)
of the startpoint and has the consensus sequence TATAAT, this sequence is
commonly known as Pribnow box . This sequence is A.T -rich and as a result,
requires the minimum energy for strand separation or ‘melting’ during initiation
of transcription.
The -35 Sequence
The midpoint of -35 sequence is, on an average, 35 bp upstream of the
startpoint, it has the consensus sequence TTGACA. This sequence plays an
important role in promoter recognition by RNA polymerase.

5’……………TTGACA…..….16-18 bp…..…..TATAAT……….…Purine………...…..3’
- 35 region
[Recognition Domain]
-10 region
[Pribnow Box]
[Unwinding Domain]
+ 1
[Start site]
The UP Element
It is an A-T-rich region located upstream of the -35 sequence. It interacts with the α-
subunit of RNA polymerase, and is found in promoters of such genes that are highly
expressed e.g. rRNA genes.
The Distance between -10 and -35 Sequences
In 90% of promoters, the distance between -10 and -35 sequences is 16-18 bp this
distance seems to be critical for proper orientation of RNA polymerase during transcription
initiation.
Constitution of a typical bacterial promoter

Bacterial RNA Polymerase
In most bacteria, a single type of RNA polymerase is found. The complete RNA
polymers molecule is called holoenzyme , its has the following components:
(1) the core enzyme (2) the sigma (σ) factor.
The holoenzyme may be symbolized as α2ββꞌσ
Core Enzyme
The core enzyme can transcribe a DNA duplex after transcription has been initiated but
it can not initiate transcription at proper sites. The core enzyme has four polypeptides
as follows: two α polypeptides, one β polypeptide and one βꞌ polypeptide. Therefore, a
core enzyme is symbolized as α2 ββꞌ.
The α Subunit
Two copies of α polypeptides are found in each molecule of core enzyme. This
polypeptide is encoded by gene rpoA. This subunit is required for assembly of the core
enzyme, it also plays a role in promoter recognition.

The β and βꞌ Subunits
These subunits are present in one copy per core enzyme molecule, and are encoded by
the genes rpoB and rpoC, respectively. The two subunits together form the catalytic
centre, and they contact DNA at many points downstream of the active transcription
site i.e., where the RNA chain is being synthesized.

The core enzyme carries out the following four functions, each function being performed
presumably by a different site :
1. DNA unwinding site unwinds the DNA duplex as it moves along DNA being transcribed.
2. The site binding to antisense strand, the strand being transcribed.
3. The site that binds to the sense strand ; this allows the antisense strand to remain single-
stranded.
4. After a segment has been transcribed, the DNA rewinding site of core enzyme is
concerned with rewinding of the sense and antisense strands into a normal duplex.
Sigma Factor
The sigma factor is involved in stable binding of RNA polymerase holoenzyme specifically
to promoter DNA. The chief function of σ factor is to ensure that the holoenzyme binds
stably at only promoter sequences. In contrast, the core enzyme binds to any DNA sequence.

Transcription Process
Transcription begins with the attachment of RNA polymerase holoenzyme to the promoter
of a transcription unit and it ends when the core enzyme reaches the terminator site and
dissociates from the DNA .
Transcription Initiation
 The initiation phase begins with the binding of RNA polymerase holoenzyme to the
promoter and ends when the holoenzyme leaves the promoter.
 The initially the holoenzyme binds to the promoter DNA about 70-80 bp extending from
-55 to +20
 The holoenzyme now induces ‘melting’, i.e. strand separation, in a <17 bp region that
includes the right end of the -10 sequence and extends just beyond the startpoint.
The transcription process is divided into the following steps:
(1) initiation (2) elongation (3) termination.

The holoenzyme now begins to transcribe the antisense strand, RNA synthesis begins
at the startpoint.
The nucleotides used are riboside 5’-triphosphates and RNA synthesis progresses in 5ꞌ
-3ꞌ direction. The enzyme permits the correct ribotides to align opposite the
deoxyribotides of antisense strand.
When the first two ribotides have been aligned, the enzyme catalyzes the formation of
phosphodiester bond between them, the diribotide so formed remains associated with
the template DNA strand and the enzyme. The holoenzyme sequentially adds the
subsequent ribotides to the growing RNA chain.
The RNA chain remains as RNA∙DNA hybrid for ~2 to 3 nucleotides at its 3-end, i.e.,
the growing end , But ~25 nucleotide at the growing end of the RNA chain are
associated with the template DNA and /or the enzyme.

Elongation
 The elongation phase begins when the RNA polymerase leaves the promoter region and
continues transcription of the template strand.
 As the core enzyme moves along the DNA duplex, the regions downstream of the
startpoint progressively become single-stranded, the core enzyme sequentially adds the
correct ribotides to the 3ꞌ-end of RNA chain.
 As the RNA chain elongates, its 5ꞌ-region progressively separates from the template
strand. The sense and antisense strands of the DNA become progressively free as the
transcription proceeds; these single strands progressively reassociate to form double
helix.
 Thus elongation involves progressive disruption of the double helix to generate transient
single-stranded regions; the separated strands form a transcription bubble within which
the template strand is transcribed. The transcription bubble moves progressively
downstream of the startpoint and disappears when transcription terminates.

Termination
When the core enzyme reaches terminator site,
(1) no further ribotides are added to the RNA chain,
(2) the RNA chain dissociates from the template strand of DNA, and
(3) the separated DNA strands reassociate to form a double helix. As a result, the
transcription bubble disappears, and
(4) the core enzyme dissociates from DNA. These events constitute the termination
phase of transcription.
The termination sites in prokaryotes have been classified into the following two groups:
(1) rho – independent terminators,
(2) rho – dependent terminators.

Rho-Dependent Terminators
 At these terminators, a polypeptide called rho-factor is required for transcription
termination.
 Most likely, rho-factor binds the RNA transcript at a recognition site (-50-90 bases
long) that is located upstream of the termination site.
 Rho-factor moves along the RNA transcript at a faster rate than dose RNA polymerase;
if it catches up with the core enzyme at the termination site, it interacts with the enzyme
to cause termination.
Rho-Independent Terminator
 Terminator of transcription at these termination sites does not require rho factor;
therefore, they are called intrinsic terminators.
 The RNA transcript from such terminators forms a typical hairpin loop, which is
followed by a run of – 6U residues.
 The poly – U region probably signals the core enzyme to leave the DNA duplex.
 The actual termination may take place at any one of several positions toward the end of
poly-U region.

RNA Polymerase
All eukaryotes possess three type of RNA polymerases called RNA polymerase I,II and III .
RNA polymerase I is located in nucleolus, and is responsible for transcription of genes for
rRNA, it is responsible for 50-70% of the activity in eukaryotes.
RNA polymerase II is located in the nucleoplasm, constitutes 20-40% of total activity and
transcribes all the genes that produce mRNA.
RNA polymerase III also occurs in nucleoplasm, provides ~10% of total polymerase
activity, and transcribes tRNA and other small RNA genes.
Transcription Factors
Transcription Factors are those points that are essential for transcription initiation, but they
are not a part of RNA polymerases. A large number of transcription factors function with
RNA polymerase II, they are divided into the following three groups: (1) basal, (2) upstream
and (3) regulatory transcription factors.
Transcription in Eukaryotes

Basal Transcription Factors
These factors are required for transcription initiation at all the promoters. They join RNA
polymerase II to form a complex around the startpoint, and determine the site of transcription
initiation. The different basal factors are follows:
(1) TFIIA, (2) TFIIB, (3) TFIID, (4) TFIIE, (5) TFIIF, (6) TFIIH, and (7) TFIIJ.
Upstream transcription Factors
These transcription factors are found in all cell types and bind to specific short sequences located
upstream of the startpoint. These factors act on any promoter having the appropriate sequence and
increase the frequency of initiation. Protein SPI is an example of upstream transcription factors.
Regulatory Transcription Factors
These factors function just like upstream factors, by they have a regulatory role. They are produced
or activated at specific times or in specific tissues. As a result they control transcription of the
concerned genes.
Promoters
Eukaryotic promoters are defined as regions that can support transcription at normal efficiency and
with the proper control. The organization of promoters for the three types of RNA polymerases differ
markedly. The promoters for RNA polymerase II usually have the following modules or functional
sequence

Initiator (Inr)
It is the region that contains startpoint and has the general form Py2CAPy5. It is recognized by RNA
polymerase II. The choice of startpoint seems to depend on the location of TATA box.
TATA Box
It is a consensus sequence of 8 bases (TATAAAAA), having only A.T base pairs, and is the only
consensus sequence that occupies a fixed position in the promoter at -25. It is usually surrounded by a
G.C-rich sequence. A minority of promoters do not a TATA box; in the case of such TATA-less
promoters, a DPE (downstream promoter element) sequence located between +28 and +32 is used in
conjunction with Inr.
CAAT Box
This sequence is located ~80 bp upstream of the startpoint, has the consensus sequence
GGCCAATCT, and it increases promoter strength.
GC Box
It has the consensus sequence GGGCGG, is usually located at -90 and may be present in several copies
in a promoter
Octamer Sequence
It has the consensus sequence ATTTGCAT and is recognized by more than one transcription factor, e.g.,
Oct-1 and Oct-2.

Sequences Inr and TATA box constitute
the core promoter to which the basal
transcription factors bind to form the
initiation complex.
Enhancer
An enhancer can stimulate any promoter
that is placed in its vicinity. The essential
role of enhancer seems to be to increase
the concentration of some of the
transcription factors in the vicinity of the
promoter, this they achieve by binding to
these factors.

The process of transcription
In eukaryotes transcription unit generally contains a single gene. Transcription termination
occurs beyond the end of coding region. Transcription by RNA polymerase II is briefly
described below.
Transcription Complex
The complex of proteins formed at the promoter that ultimately leads to transcription
initiation is called transcription complex.

Transcription Initiation
 Transcription begins at the startpoint, which is usually an A.
 A closed binary complex is formed when RNA polymerase II binds the promoter.
 The complex is then converted into an open binary complex in which the two strands of
promoter DNA become separated locally.
 RNA polymerase now begins transcription at the startpoint, and presumably, the
transcription factors are released around this stage.
 The basal transcription factors are able to initiate transcription at a low level.
 Efficient initiation requires the effects of certain upstream and/or regulatory factors;
these factors most likely interact with the basal factors and, thereby, enhance the
frequency of formation of initiation complex.
Transcription Termination
 It is likely that the transcription units have rather long terminator regions with multiple
terminator sites.
 In the virus SV40, the termination site consists of a hairpin loop, followed by a stretch of
U bases; this is similar to the intrinsic terminators of prokaryotes.
 In some transcription units, termination occurs 1,000 bp downstream of the site
corresponding to the 3' -end of mature mRNAs. In such cases, the 3' -ends of mRNAs are
generated by cleavage following transcription.

mRNA Processing
 The process of modification, mainly through cleavage and /or splicing of primary RNA
transcripts so as to produce functional mRNA molecules from them is called RNA
processing.
 RNA processing is carried out by ribonucleases, the enzymes that cleave RNA.
 They are of two types viz., Exoribonucleases and Endoribonucleases.
 Exoribonucleases remove one base at a time from one end of RNA molecule, all know
enzymes trim RNA from the 3' end But endoribonucleases produce internal cuts in RNA
molecules.
 Eukaryotic transcripts (pre-mRNA) contains exons (coding/expressed sequences) and
introns (non coding/ unexpressed sequences)
 Post-transcriptional modifications (i.e. splicing) remove introns before shipping the final
mRNA to the cytoplasm.
 All the introns are excised from the primary transcript and all the
exons of a gene are joined together in the proper order; this process is called splicing.
Splicing can occur in the following three ways.
 Spliceosome – Mediated splicing
 Alternative Splicing
 Alteration of mRNA Ends

Spliceosome – Mediated splicing
 Spliceosomes are organelles in which the
excision and splicing reactions that remove
introns from pre-mRNA occur.
 Spliceosomes are ellipsoid particles of RNA and
protein, each spliceosome is ~25x50 nm in size.
 Spliceosomes are assembled on precursor RNA
molecules by an association of four different
small nuclear ribonucleoprotein particles
(snRNPs) and 40 different proteins often called
splicing factors.
 The snRNPs involved in spliceosome formation
are snRNP U1, snRNP U2, snRNP U5 and
snRNP U4/U6. Each snRNP has a single RNA
molecule and 10 different proteins, thus the 4
snRNPs in a spliceosome contain 40 different
proteins

Alternative Splicing
 When a single pre-mRNA molecule (primary transcript) is processed in two or more
different ways to yield two or more different types of mRNAs, it is called alternative
splicing.
 The resulting mRNAs encode different forms of the protein, known as isoforms. An
example of alternative splicing is provided by splicing in male and female drosophila of
the pre-mRNA produced by tra gene.
 It is estimated that 60% of all human genes are expressed as alternatively spliced mRNAs
 Alternative splicing is illustrated below for the fibronectin gene. The fibroblast and
hepatocyte isoforms differ in their content of the EIIIA and EIIIB domains which mediate
cell surface binding. Twenty different isoforms of fibronectin produced by alternative
splicing have been identified

Alteration of mRNA ends
Each end of a pre-mRNA molecule is modified in a particular way
 The 5 end receives a modified nucleotide cap (RNA capping).
 The 3 end gets a poly-A tail (Polyadenylation).
RNA capping happens at the 5 end of the RNA, usually adds a methylgaunosine shortly
after RNA polymerase makes the 5end of the primary transcript
Polyadenylation modifies the 3end of the primary transcript by the addition of a string of A’s.

Polyadenylation
 mRNAs are also modified at the 3' end
by polyadenylation.
 This involves cleavage of the longer pre-
mRNA at the polyadenylation site and
the addition of up to 250 adenylate
residues by template-independent
poly(A) polymerase.
 Non-coding RNA intron sequences are
excised and the coding exon sequences
are ligated to form the functional mRNA
by the process known as splicing.
 The mRNA retains 5‘ and 3'
untranslated regions (UTRs) at each
end.
 The poly(A) tail helps protect the
mRNA.

Capping
 Transcription usually begins with a purine (A or G)
triphosphate, the P is located at position 5 of this A
or G.
 Soon after the transcription begins a guanine
triphosphate is added to the 5 -end of the RNA, this
G forms a 5-5 bond with the terminal A or G of the
RNA and the bond is mediated by 3 phosphate
residues. This G is known as 5-CAP the process is
called capping and the enzyme involved is guanyl
transferase.
 Subsequently the 5-cap may also be methylated.
 All mRNAs of eukaryotes are capped.
 The 5' cap is important for transport of the mRNA to
the cytoplasm, protection against nuclease
degradation, and initiation of translation

Translation
 The mRNA carries genetic information encoded as a ribonucleotide sequence from
the chromosomes to the ribosomes.
 The ribonucleotides are "read" by translational machinery in a sequence of nucleotide
triplets called codons. Each of those triplets codes for a specific amino acid.
 Translation is the production of proteins by decoding mRNA produced in transcription.
 The ribosome and tRNA molecules translate this code to produce proteins.
 The ribosome is a multi subunit structure containing rRNA and proteins. It is the "factory"
where amino acids are assembled into proteins.
The process of translation requires the following major components:
 mRNA
 ribosome (containing rRNA)
 tRNA
 translation factors

 is a RNA that carry the information (or message) that is encoded in the genes to the
sites of protein synthesis in the cell,
 single-stranded macromolecule,
 synthesized during transcription,
 In eukaryotic cells, once mRNA has been transcribed from DNA, it is
"processed" before being exported from the nucleus into the cytoplasm, where it is
bound to ribosomes and translated into its corresponding protein with the help of tRNA.
 In prokaryotic cells, which have no partition into nucleus and cytoplasm compartments,
mRNA can bind to ribosomes while it is being transcribed from DNA.
 In prokaryotes mRNA molecule codes for more than one polypeptide. Such an mRNA
is known as polycistronic mRNA.
 in eukaryotes, a single mRNA encodes for only one polypeptide chain. Such an mRNA
is known as monocistronic mRNA.
Messenger RNA

39
Messenger RNA (mRNA)
methionine glycine serine isoleucine glycine alanine stop
codon
protein
A U G G G C U C C A U C G G C G C A U A A
mRNA
start
codon
Primary structure of a protein
aa1 aa2 aa3 aa4 aa5 aa6
peptide bonds
codon 2 codon 3 codon 4 codon 5 codon 6 codon 7codon 1

All mRNA molecules have a translation initiation site close to their 5ꞌ-end and a chain
termination site towards the 3ꞌ-end. The chain initiation site consists of the codon AUG. The
chain termination site has one or two of the following three nonsense codons; UAA, UAG
and UGA.
Both prokaryotic and eukaryotic mRNAs have the following regions:
I. A 5ꞌ leader sequence that is not translated,
II. The coding region, which begins with a translation initiation codon (ordinarily, AUG)
and ends with a translation termination codon,
III. A nontranslated 3ꞌ trailer.
Translation initiation site
 In prokaryotes, the initiation site has usually the codon AUG.
 The 5ꞌ leader of bacterial mRNAs has a consensus hexamer sequence called Shine-
Dalgarno sequence (5ꞌ AGGAGG 3ꞌ), located 7 bases upstream of the AUG (initiation)
codon.
The bacterial 16 S rRNA has near its 3ꞌ-end a highly conserved hexamer sequence
3ꞌ UCCUCC5ꞌ.
This sequence is complementary to and base- pairs with the Shine-Dalgarno sequence of
mRNA, this base pairing allows the smaller subunit of ribosomes to bind mRNA during
translation.

 In eukaryotes, some cap-binding
proteins recognise and bind to the 5ꞌ-
cap of mRNA. The smaller (40 S)
subunit of the ribosome now binds
the 5ꞌ-cap.
 In some mRNAs, the initiation
codon, AUG, lies within 40 bases of
the cap.
 In such cases, the 40S subunit
moves from the 5’-cap till it reaches
the AUG codon.
 The AUG codon functions most
efficiently in translation initiation
when it is part of the following
sequence GCCA (or G) CCAUGG .
Termination site
The termination site is marked by one of
the three nonsense codons, viz., UAA,
UAG and UGA; these codons do not
code for any amino acid. Translation is
terminated when the ribosome reaches
the termination site.

The genetic code
 The number and the sequence of bases in mRNA specifying an amino acid is known as
codon.
 The set of bases in a tRNA that base-pairs with a codon of an mRNA is known as
anticodon.
 The sequence of bases in an anticodon is exactly the opposite of and complementary to
that present in the codon. For example the codon 5’ AUG 3’ has the anticodon 3’ UAC
5’.
 The set of all the codons that specify the 20 amino acids is termed as the genetic code,
genetic language or coding dictionary.
Properties of the genetic code:
 The code is universal: All prokaryotic and eukaryotic organisms use the same codon to
specify each amino acid.
 The code is triplet: Three nucleotides make one codon. 61 of them code for amino acids
and 3 viz. , UAA, UAG and UGA are nonsense codons or chain termination codons.

The code is degenerate: There are 64 codons available for 20 amino acids. Most amino
acids are encoded by two or more codons
The code is non ambiguous: Each codon specifies only one of the 20 amino acids. None
of the codons code for two or more amino acids. The only exception is the AUG codon
which codes for formylmethionine in prokaryotes at the initiation site. While at other
positions it specifies methionine.
The code is non overlapping: A base in mRNA is not used for two different codons.
The code is commaless: There is no special signal or commas between codons.
Wobble Hypothesis
Wobble hypothesis was proposed by Crick in 1966. According to it the first two bases of a
codon pair strictly according to the normal base pairing rules with the last two bases of the
anticodon. This seems the possible explanation for degeneracy of codons.

Amino acids encoded by the 64 possible codons of the
triplet code.

Ribosomes And Ribosomal RNA
 Ribosomal RNA (rRNA) occurs in association with proteins and is organized into almost spherical
bodies of about 200 A° diameter called ribosomes.
 Ribosomes of prokaryotes are of 70 S size and are composed of 60% rRNA and 40% protein. They
dissociate into a smaller 30 S subunit and a larger 50 S subunit.
 The 30S subunit of prokaryotic ribosomes has a single 16S rRNA molecule, which is associated with
21 different ‘S’ proteins. The larger 50S subunit has a 23S rRNA and a 5S rRNA molecule
complexed with 31 different ‘L’ proteins. The basic structure of ribosomes is due to their rRNA, and
the proteins play a secondary role.
 The cytoplasmic ribosomes of eukaryotes are of the size 80S and contain 40% rRNA and 60%
protein. They consist of a 40S and a 60S subunit.
 The 40 S subunit of eukaryotic ribosomes has a single 18S rRNA molecule and 33 different proteins.
The larger 60S subunit has one molecule each of 28S, 5.8S and 5S rRNA and 49 different proteins.
 The larger subunit of the ribosomes serves to stabilize the amino acid-bound tRNA, while the
smaller subunit stabilizes the mRNA. This allows the formation of peptitide bonds between the two
amino acids attached to the tRNA molecules base-paired with the two adjacent codons on mRNA.

Functional sites on Ribosomes
 A complete ribosome particle has two distinct and adjacent sites for the attachment of
aminoacyl-tRNAs (amino acid carrying tRNA), an aminoacyl attachment site (site A)
and a peptidyl site (site P).
 An aminoacyl-tRNA first attaches to site A, the kind of aminoacyl-tRNA being
determined by the sequence of mRNA (codon) attached to the site A. the aminoacyl-
tRNA, along with the mRNA codon, now moves to the site P, making the site A available
for the attachment of a new aminoacyl-tRNA.
 In addition, the ribosome has following sites as well:
1.mRNA site 2.peptidyl transferase site 3.EF-Tu site, 4.EF-G site
5. 5 S RNA site 6.exit site.

 Transfer RNA (tRNA) is a class of RNA of small size of 3S and generally, it has 76 to 95
nucleotides.
 tRNA molecules have a high proportion of unusual bases, viz., dihydrouridine (Dih U),
pseudouridine(Ψ), inosine (I), methylated bases and thymine (T). The unusual bases
present in tRNA are produced after transcription by modifications of the usual bases, viz.,
A,U,G and C.
 Further, tRNA molecules show considerable double helical structure, and more than 50%
of the bases in tRNA are paired.
 The existence of tRNA was demonstrated by Hoagland and coworkers in 1957.
Structure of tRNA
 A ‘clover leaf model’ of tRNA structure has been proposed to account for its structural and
functional properties.
 The three nucleotides at the 5 -end are CCA in all the types of tRNA molecules. This
segment is the amino acid acceptor region. The amino acid binds to one of the OH groups
of the ribose of the terminal adenine.
 The tRNA molecule has three loops: DHU loop, anticodon loop and thymine loop.
 Sometimes an extra loop, having a variable number of nucleotides, is also present between
the anticodon and thymine loops.
Transfer RNA

The anticodon loop is the second
from the 5 -end, has 7 unpaired
bases and is located opposite to the
amino acid acceptor region(CCA). It
has 3 nucleotides that function as the
anticodon. The distance of anticodon
from the 3ꞌ acceptor end is uniformly
66A°.
The thymine loop is located near the
3-end, has 7 unpaired bases and is
likely to function as the ribosome
attachment region.
The DHU loop has 8-12 unpaired
bases and serves as the aminoacyl
synthetase recognition region.

Formation of aminoacyl-tRNA
 The amino acids are attached to tRNA molecules through a two step reaction process. Both
the reactions are catalysed by the same enzyme, aminoacyl tRNA synthetase.
 The carboxyl group of the amino acid reacts with one of the –OH groups of the ribose of
the terminal adenine nucleotide to produce aminoacyl-tRNA .
1.first step: It consist of amino acid activation, in which the amino acid molecule reacts
with and ATP molecule to yield an aminoacyl-AMP (aminoacyl adenylate) molecule.
2.Second step: The amino acid form aminoacyl-AMP molecule is then transferred to a
tRNA molecule that is specific for the amino acid, and AMP is released.
 The aminoacyl-AMP complex remains tightly bound to the enzyme aminoacyl-tRNA
synthetase during the entire reaction, i.e., both the steps.
 The attachment of an amino acid to the concerned tRNA to yield aminoacyl-tRNA is
called charging of tRNA.
Recognition of codons
 Recognition of specific codons of the mRNA is the function of tRNA molecule. This
recognition is due to the anticodon that base pairs with the mRNA codon.
 Almost as a rule, A in the first place of anticodons (corresponding to the third base of
mRNA codon) is modified to I (inosine); I base pairs with any one of U,C and A.
 Similarly, U in the first position of anticodon is always modified the modified U may base
pair with A,G and, in some cases with even U.

The translation process
Translation begins near the 5-end of mRNA and progresses toward its 3 -end. In terms of the
polypeptide being synthesized, it starts with the N-terminal end of the polypeptide, while the
C-terminal amino acid is the last to be added to the chain.
The process of translation may be divided into the following 3 steps:
(1) initiation,
(2) elongation and
(3) termination.
Initiation
Initiation comprises all the events that precede the formation of the first peptide bond. It
includes the following events:
(1) binding of the smaller subunit of ribosome to mRNA and
(2) binding of the first or initiator aminoacyl-tRNA to the P site of ribosome; this completes
the formation of initiation complex. Initiation is a relatively slow step in translation.

Initiation in prokaryotes
The formation of initiation complex in E. coli occurs as follows.
1. Initiation factor IF3 binds to a free 30S subunit of ribosome, now this subunit can not
associate with a 50S subunit.
2. The 30 S subunit bound to IF3 now binds to the ribosome binding site or Shine-
Dalgarno sequence of mRNA. IF3 is absolutely necessary for this binding.
3. IF1 binds to 30 S subunit bound to mRNA and possibly stabilizes the initiation complex.
4. The 30S-IF3 complex ultimately positions itself on the mRNA in such a way that the
initiation codon AUG is located in the P (and not the A) site of the 30S subunit.
5. The formylmethionyl-tRNAf (fmet-tRNAf ) binds to IF2 to form a binary complex. The
binary complex along with one molecule of GTP binds the codon AUG in the P site.
Only fmet-tRNAf can enter the P site; all other tRNAs can not gain a direct access to the
P site.
6. Factor IF1 binds to the A site as a part of the complete initiation complex. This prevents
aminoacyl-tRNA from entering the A site.
7. The 50 S subunit of ribosome now joins the 30S subunit; GTP is hydrolysed and IF2 and
IF3 are released. The initiation complex is now complete with an active ribosome in
which A site is vacant while the P site is occupied by fmet-tRNAf.

GTPInitiator tRNA
mRNA
5
3
mRNA binding site
Small
ribosomal
subunit
Start codon
P site
5 3
Translation initiation complex
E A
Large
ribosomal
subunit
GDP
The initiation process in Prokaryotes involves first joining the mRNA, the
initiator methionine-tRNA, and the small ribosomal subunit. Several
“initiation factors”--additional proteins--are also involved. The large
ribosomal subunit then joins the complex.

Initiation in eukaryotes
Initiation in eukaryotes requires at least nine different factors. It proceeds as follows.
1. First of all initiation factor eIF4F (e prefix denotes eukaryotic) binds to the 5 -cap of
mRNA. eIF4F consists of eIF4F, eIF4E and eI4G. eIf4E binds to the 5 -cap, while eIF4A
(a helicase) and eIF4B together unwind any secondary structure that may be present in the
first 15 bases of mRNA.
2. The initiator aminoacyl-tRNA, viz., Met-tRNAi (methionyl – tRNAi) binds to eIF2- GTP
complex.
3. The Met-tRNAi-eIF2-GTP complex now binds in the P site of a free 40S subunit of the
ribosome.
4. This 50S subunit of ribosome now binds to the 5 -cap of mRNA; it then moves along
RNA till it reaches the initiation codon AUG. this process required eIF3 and several other
factors like eIF4, eIF4 A and eLF4B.
5. Factor eIF6 binds to the 60 S subunit and prevents its association with a free 40S
subunit. Now 60S-eIF6 complex joins the 40 S subunit bound to mRNA; both eIF2 and
eLF3 are released due to the action of eLF5. The joining of 40S and 60S subunits is
mediated by eLF4 C.
6. Once the complete 80 S ribosome is assembled at the AUG codon, probably all the
remaining initiation factors are released.

Elongation
 Elongation includes all the reactions from the formation of the first peptide bond to that
of the last peptide bond of the polypeptide chain.
 Amino acids are added one at a time to the growing peptide chain.
 It is the most rapid step of translation: in bacteria it proceeds at the rate of 15 amino
acids/second at 370C, while in eukaryotes its rate is 2 amino acids/second.
The elongation step in bacteria is briefly described below. The complete initiation
complex has the following features:
 ribosome is complete and ready for translation,
 P site of ribosome is occupied by the initiator aminoacyl-tRNA (fmet-tRNAf) and
 The A site is free.
 The P site of ribosome must be occupied by peptidyl-tRNA (tRNA carrying a peptide or
fmet-tRNA for the A site to be in a proper conformation to allow entry to an amino-acyl-
tRNA.
 Any amino-acyl-tRNA (except fmet-tRNA) can enter the free A site of an active
ribosome, depending mainly on the codon of mRNA present at the A site.

Elongation
Elongation is divided into three steps:
1. Aminoacyl tRNA delivery:
 EF-Tu is required to deliver the aminoacyl tRNA to the A site and energy is consumed in
this step by the hydrolysis of GTP.
 The released EF-Tu. GDP complex is regenerated with the help of EF-Ts.
 In the EF-Tu.EF-Ts exchange cycle EF-Ts displaces the GDP and subsequently is
displaced itself by GTP.
 The resultant EF-Tu. GTP complex is now able to bind another aminoacyl tRNA and
deliver it to the ribosome.
 All aminoacyl tRNAs can form this complex with EF-Tu except the initiator tRNA.
2. Peptide bond formation.
 After aminoacyl -tRNA delivery, the A- and P- sites are both occupied and the two amino
acids that are to be joined are in close proximity.
 The peptidyl transferase activity of the 50S subunit can now form a peptide bond
between the two amino acids without the input of any more energy, since energy in the
form of ATP was used to charge the tRNA.
 The cycle is repeated until one of the termination codons (UAA, UAG and UGA) appear
in the A-site.

3.Translocation.
 After peptide bond formation the P site has a free tRNA, while the A site has a peptidyl-
tRNA.
 Now the ribosome moves 3 bases along the mRNA , this removes the free tRNA from
the P site and moves the peptidyl tRNA from A site into the now free P site.
 In prokaryotes the free tRNA leaves the ribosome via the exist site, while in eukaryotes
it directly goes into the cytosol. This whole process if called translocation .
 It requires elongation factor EF-G and GTP.
 EF-G-GTP complex binds to the EF-G site of 50 S subunit, GTP is hydrolysed the
ribosome moves 3 bases along the mRNA and EF-G and GDP are released from the
ribosome
 Release of EF-G is essential to enable binding of EF-Tu to the A site.
 Translocation generate a free A site that can bind to the appropriate ternary complex. In
this manner, ribosome moves 3 bases at a time and one amino acid is added after every
translocation event.
 In case of eukaryotes, factor eEF2 functions in the translocation as does EF-G in
bacteria.

Termination
 Termination describes those events that occur after the formation of the last peptide
bond of the polypeptide, they culminate in the separation of the ribosome from mRNA.
 Termination is signaled by the nonsense codons UAG, UAA and UGA, and is mediated
by certain proteins called release factors.
 E. coli has two release factors, RF1 and RF2.
 Factor RF1 recognizes UAA and UAG while RF2 recognizes UGA and UAA.
 When the A site of a ribosome reaches a nonsense codon the specific release factor (RF1
or RF2) enters A site, this requires the P site to be occupied by peptidyl-tRNA.
 As a result of this the following three simultaneous events occur:
(1)the polypeptide chain detaches from the tRNA located at P site,
(2) followed by immediate release of tRNA from the P site and
(3) the release of ribosome from the mRNA.
 The two subunits of ribosomes may dissociate after their release.
 The RF1 or RF2 itself is released from ribosomes by the action of RF3.
 In case of eukaryotes, eRF1 recognizes and binds the termination codons. Factor eRF1
is released from ribosomes by eRF3.

59
End Product –The Protein!
• The end products of protein synthesis is a
primary structure of a protein
• A sequence of amino acid bonded together by
peptide bonds
aa1
aa2 aa3 aa4
aa5
aa200
aa199

Conclusion
 Genetic information is encoded in the base sequence of DNA molecules as a series of
genes.
 Gene expression is the term used to describe how cells decode the information to
synthesize proteins required for cellular function.
 The expression of a gene involves the synthesis of a complementary RNA molecule whose
sequence specifies the amino acid sequence of a protein.
 The DNA sequence of the gene is collinear with the amino acid sequence of the
polypeptide.
 Amino acids are encoded by 64 base triplets called codons which encode the 20 amino
acids.
 Most amino acids have more than one codon. This is known as the degeneracy of the
genetic code and it helps to minimize the effect of mutations.

Codons that specify the same amino acid are known as synonyms and differ at
their third base, known as wobble position.
AUG is the initiation codon and encodes methionine. There are three stop codons:
UAG, UGA and UAA.
An open reading frame is a sequence of codons, bounded by start codons and stop
codons.
The genetic code applies universally with all organisms using the same codons for
each amino acid.
Translation is similar in prokaryotes and eukaryotes and occurs in three stages
(initiation, elongation and termination). Each stage involves a set of accessory
proteins.
Energy is provided by hydrolysis of Adenine triphosphate and Guanosine
triphosphate.

References
 Crick, F.H.C. On Protein Synthesis, Medical Research Council Unit for the study
of Molecular Biology, Cavendish Laboratory, Cambridge. Pp: 138-163
 Singh, B.D. Fundamentals of Genetics, Kalyani publications, Pp: 524-566.
 www.wikipedia.com

Role of DNA and RNA in Protein Synthesis

Role of DNA and RNA in Protein Synthesis

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