Transcription & Translation

DNA RNA Protein

• Dr Bibekanand Mallick
• Associate Professor
• Department of Life Science
• NIT Rourkela
 Transcription
This is very similar to DNA replication but there
are some important differences:
1. RNA is made of ribonucleotides
2. RNA polymerase catalyzes the reaction
3. The synthesized RNA does not remain base-paired to
the template DNA strand
4. Less accurate (error rate: 10-4)
5. Transcription selectively copies only certain parts of
the genome and makes one to several hundred, or
even thousand, copies of any given section of the
genome. (Replication?)
Key enzyme:
RNA polymerases come in different forms,
but share many features
• RNA polymerases perform essentially the same
reaction in all cells
• Bacteria have only a single RNA polymerase, while in
eukaryotes have three: RNA Pol I, II and III

• RNA Pol II is the focus of eukaryotic transcription,

because it is the most studied polymerase, and is also
responsible for transcribing most genes-indeed,
essentially all protein-encoding genes
• RNA Pol I transcribe the large ribosomal RNA
precursor gene
• RNA Pol III transcribe tRNA gene, some small nuclear
RNA genes and the 5S rRNA genes
The subunits of RNA polymerases
“Crab claw” shape of RNAP”
(The shape of DNA pol is__)

Active center cleft

There are various channels allowing DNA, RNA and

ribonucleotides (rNTPs) into and out of the enzyme’s
active center cleft
Transcription by RNA polymerase proceeds
in a series of steps:

Initiation
Elongation
Termination
 Initiation
Consensus sequence of
the -35 and -10 region
 Promoter: the DNA sequence
that initially binds the RNA
polymerase
 -10 region is called Pribnow Box
 The sigma factor mediates
binding of polymerase to the
promoter
 The structure of promoter-
polymerase complex undergoes
structural changes to proceed
transcription
 DNA at the transcription site
unwinds and a “bubble” form
 Direction of RNA synthesis
occurs in a 5’-3’ direction (3’-end
growing)
Transcription initiation involves 3 defined
steps:

1. Forming closed complex

2. Forming open complex
3. Promoter escape
 Forming Closed complex
• The initial binding of polymerase to a promoter
• DNA remains double stranded
• The enzyme is bound to one face of the helix
Sigma factor:
Region 4 recognizes -35 element of promoter
Region 2 recognizes -10 element
Region 3 recognizes the extended –10 element

Forming Open complex

• The DNA strand separate over a distance of ~14 bp (-11 to
+3 ) around the start site (+1 site). This is called ‘melting’
• Transcription bubble forms

Promoter escape
• The enzyme escapes from the promoter
• The transition to the elongation phase
• Forming a stable ternary complex =DNA +RNA + enzyme
Elongation
 Once the RNA polymerase
has synthesized a short
stretch of RNA (~ 10 nt),
transcription shifts into the
elongation phase.
 This transition requires
further conformational
change in polymerase that
leads it to grip the template
more firmly.
 Functions: synthesis RNA,
unwinds the DNA in front,
re-anneals it behind,
dissociates the growing
RNA chain
 Termination
• After the polymerase transcribes the length of the gene (or
genes), it will stop and release the RNA transcript.
• In some cells, termination occurs at the specific and well-
defined DNA sequences called terminators. Some cells lack
such termination sequences.
Two types of termination:
Rho-dependent:
 Rho, a ring-shaped protein binds to newly synthesized RNA and terminates
transcription.
 Induces a conformational change in polymerase, causing RNA polymerase to
dissociate from DNA for transcription termination
Rho-independent:
 Does not require other factors, rather depends on terminator sequence.
 The terminator sequence is made up of a short-inverted repeat (~20 nts)
followed by a stretch eight A:T base pairs. When polymerase transcribes an
inverted repeat sequence, a hairpin is formed in the resulting RNA which
induces termination by either pushing polymerase forward relative to the DNA
and RNA or inducing a conformational change in polymerase.
 TRANSLATION
Proteins are made using mRNA as recipe

Ribosomes are the protein-synthesizing machines of the cell.

They translate the information encoded in messenger RNA (mRNA)

into a polypeptide
Subunits
A ribosome is made from complexes of RNAs and proteins and is
therefore a ribonucleoprotein..
 TheAgenetic
Codon: codon iscode
a sequence of three nucleotides that corresponds with a
specific amino acid or stop signal during protein synthesis.
 Charging tRNA (Adding amino acid to tRNA)
• Aminoacyl tRNA synthetases:
catalyzes the attachment of a specific
amino acid to its corresponding tRNA.
The charging process (aminoacylation)
produces a charged tRNA (aminoacyl-
tRNA), using energy from ATP
hydrolysis.
•There are 20 different aminoacyl-
tRNA synthetase enzymes, one for
each amino acid. Some of these
enzymes recognize tRNAs by their
anticodon regions.
• amino acid + tRNA + ATP →
aminoacyl-tRNA + AMP + Ppi
• The tRNA binds to the enzyme, and
the amino acid is transferred onto it,
displacing the AMP. The aminoacyl-
tRNA is released from the enzyme.
 Initiation of Translation

1. Protein synthesis is divided into three stages:

a. Initiation
b. Elongation
c. Termination

2. Initiation of translation requires:

a. An mRNA
b. A ribosome
c. A specific initiator tRNA
d. Initiation factors
e. Mg2+ (magnesium ions)
 Prokaryotic translation begins with binding of the 30S ribosomal
subunit to mRNA near the AUG codon. The 30S comes to the
mRNA bound to:
a. All three initiation factors, IF1, IF2 and IF3.
b. GTP
c. Mg2+.
 Ribosome binding to mRNA requires more than the AUG:
 An additional sequence 8–12 nucleotides upstream from the
AUG is commonly involved, named as Shine and Dalgarno
sequence.
Initiation of protein synthesis in prokaryotes
 Next, the initiator tRNA binds the AUG to which the 30S subunit
is bound. AUG universally encodes methionine. Newly made
proteins begin with Met, which is often subsequently removed.
a. Initiator methionine in prokaryotes is formylmethionine (fMet).
It is carried by a specific tRNA
b. The tRNA first binds a methionine, and then transformylase
attaches a formyl group to the methionine, making fMet-tRNA.
fMET (a charged initiator tRNA).

 When fMet-tRNA binds the 30S-mRNA complex, IF3 is released

and the 50S ribosomal subunit binds the complex. GTP is
hydrolysed, and IF1 and IF2 are released. The result is a 70S
initiation complex consisting of:
a. mRNA.
b. 70S ribosome (30S and 50S subunits) with a vacant A site.
c. fMet-tRNA in the ribosome’s P site.
The formation of a peptide bond between the first two amino acids
of a polypeptide chain is catalyzed on the ribosome by peptidyl
transferase
 Elongation
During elongation, the ribosome travels on the mRNA on each of the codon and
then, the charged tRNAs enter at the A site with the help of elongation factor EF-
Tu, then shift to the P site, and then finally to the E site for removal.
Translocation denotes the simultaneous movement of two
tRNAs together with the mRNA by one codon on the
ribosome
1. The ribosome now advances one codon along
the mRNA. EF-G (elongation factor-G) is used in
translocation in prokaryotes. EF-G-GTP binds the
ribosome, GTP is hydrolyzed and the ribosome
moves 1 codon while the uncharged tRNA leaves the
P site.
2. Release of the uncharged tRNA involves the
50S ribosomal E (for Exit) site. Binding of a charged
tRNA in the A site is blocked until the spent tRNA is
released from the E site.
3. During translocation the peptidyl-tRNA remains
attached to its codon, but is transferred from the
ribosomal A site to the P site.
4. The vacant A site now contains a new codon,
and an aminoacyl-tRNA with the correct anticodon
can enter and bind. The process repeats until a stop
codon is reached.
 Termination of Translation
1. Termination is signaled by a stop codon/ non-sense codon
(UAA, UAG, UGA), which has no corresponding tRNA.

2. Release factors (RF) assist the ribosome in recognizing the

stop codon and terminating translation.
a. In E. coli:
i. RF1 recognizes UAA and UAG.
ii. RF2 recognizes UAA and UGA.
iii. RF3 stimulates termination.

3. Termination events triggered by release factors are:

a. Peptidyl transferase releases the polypeptide from the
tRNA in the ribosomal P site.
b. The tRNA is released from the ribosome.
c. The two ribosomal subunits and RF dissociate from the
mRNA.
d. The initiator amino acid (fMet or Met) is usually cleaved
from the polypeptide.
Termination of translation