Transcription and

Translation
• Types of RNA
• Prokaryotic Transcription
• Regulation of Prokaryotic Transcription
• Eukaryotic Transcription
• Differences between Prokaryotic and Eukaryotic Transcription
• Regulation of Eukaryotic transcription
• RNA Processing
 Recap
• DNA is a double-stranded molecule.
• Each of the two DNA strands is a polymer of nucleotides linked by phosphodiester bonds.
• The two strands are held together by hydrogen bonds between nucleotides with
complementary bases (adenine–thymine and cytosine–guanine).
• A gene is a sequence of nucleotides whose base sequence encodes the amino acid
sequence of a polypeptide.
Important Features
• a. DNA contains genetic template" for proteins.

• b. DNA is found in the nucleus

• c. Protein synthesis occurs in the cytoplasm -

ribosome.

• d. "Genetic information" must be transferred to the

cytoplasm where proteins are synthesized.
Processes of Protein Synthesis
• a. Transcription - genetic template for a protein is copied and carried
out to the cytoplasm

• b. Translation - template serves as a series of codes for the amino

acid sequence of the protein
 Cells Use RNA to Make Protein
• The RNA Players – mRNA, rRNA, tRNA
• During polypeptide synthesis, ribosomal RNA (rRNA) is
the site of polypeptide assembly.
• Transfer RNA (tRNA) transports and positions amino acids.
• Messenger RNA (mRNA) directs which amino acids are
assembled into polypeptides.
• Central Dogma
• DNA  RNA Protein
Central Dogma of Gene Expression
There are Different RNAs with Distinct Functions
Introns and Exons
• In eukaryotes, each gene contains non-coding sequences of bases
(introns) and coding sequences of bases (exons).
Eukaryotic Genes are Segmented

Genes are made of parts represented in the mRNA (exons) and parts that are
transcribed but not present in the mRNA (introns).

Introns are removed from the primary transcript and exons are spliced together to
make mRNA.

In some genes more than 90% of the pre-mRNA is destroyed, never to appear in
the mRNA.
 Gene Expression
• Transcription – in the nucleus
• DNA sequence is transcribed into RNA sequence
• initiated when RNA polymerase binds to promoter
binding site
• moves along DNA strand and adds corresponding
complementary RNA nucleotide
• disengages at stop signal
 Steps of Transcription
a. DNA unwinds
b. One side of DNA "codes for a protein"
c. Genetic code of DNA is a triplet code of 3 nucleotides or bases
d. Each triplet is specific for the coding of a single amino acid
e. Sequence of triplet codes on DNA will specify the amino acid
sequence on the protein
f. Major step is the synthesis of the coded "messenger" molecule
– mRNA
g. mRNA is "transcribed" from DNA by complementary base
pairing (mRNA has no thymine, which is replaced by uracil)
h. mRNA passes out to cytoplasm to the ribosome
Transcription
• RNA polymerase
• only one of two DNA strands (template) is transcribed
• non-transcribed strand is termed coding strand - same as RNA (except T’s are
U’s)
• In both bacteria and eukaryotes, the polymerase adds ribonucleotides to the
growing 3’ end of an RNA chain.
• synthesis proceeds in 5’3’ direction
Transcription Bubble
Transcription
• Promoter
• Transcription starts at RNA polymerase binding sites called promoters on DNA
template strand.
• Initiation
• Other eukaryotic factors bind, assembling a transcription complex.
• RNA polymerase begins to unwind DNA helix.
Transcription
• Elongation
• Transcription bubble moves down DNA at constant rate leaving growing RNA
strands protruding from the bubble.
• Termination
• Stop sequences at the end of the gene cause phosphodiester bond formation
to cease, transcription bubble to dissociate, and RNA polymerase to release
DNA.
Transcription
• Eukaryotic transcription differs from prokaryotic transcription:
• three RNA polymerase enzymes
• initiation complex forms at promoter
• RNAs are modified after transcription
Segments of DNA (GENES) are the instructions that control the production of proteins.

Genetic messages can be decoded by copying part of the nucleotide sequence from
DNA into RNA. RNA contains coded information for making proteins

How does the DNA get made into RNA and that made into Protein???
Transcription in Prokaryotes

• The process of synthesis of RNA by copying the template strand of DNA is called transcription.
• During replication entire genome is copied but in transcription only the selected portion of genome is copied.
• The enzyme involved in transcription is RNA polymerase. Unlike DNA polymerase it can initiate transcription by
itself, it does not require primase. More exactly it is a DNA dependent RNA polymerase.
 The steps of transcription

• Transcription is an enzymatic process. the mechanism

of transcription completes in three major steps

1. Initiation:
closed complex formation
Open complex formation
Tertiary complex formation
2. Elongation
3. Termination:
Rho-independent
Rho- dependent
Initiation
• The transcription is initiated by RNA
polymerase holoenzyme from a specific
point called promotor sequence.
• Bacterial RNA polymerase is the principle
enzyme involved in transcription.
• Single RNA polymerase is found in a
bacteria which is called core polymerase
and it consists of α, β, β’ and ω sub units.
• The core enzyme bind to specific
sequence on template DNA strand called
promotor. The binding of core
polymerase to promotor is facilitates and
specified by sigma (σ) factor. (σ70 in case
of E. coli).
Initiation
• The core polymerase along with σ-
factor is called Holo-enzyme ie.
RNA polymerase  holoenzyme.
• In case of e. coli, promotor consists
of two conserved sequences 5’-
TTGACA-3’ at -35 element and 5’-
TATAAT-3’ at -10 element. These
sequence are upstream to the site
from which transcription begins.
Binding of holoenzyme to two
conserve sequence of promotor
form close complex.
• In some bacteria, the altered
promotor may exist which contain
UP-element and some may contain
extended -10 element rather than
-35 element.
Initiation
• Region1: it includes 1.2 and 1.1 region. Region 1.1 acts as molecular mimic of DNA
• Region2: it recognizes -10 element in promotor. α-helix recognizes -10 element.
• Region 3: it recognizes extended -10 element.
• Region 4: it recognizes -35 element in promotor by a structure called helix-turn-helix.
• The UP-element is recognized by a carboxyl terminal domain of α-sub unit called αCTD
(carboxyl terminal domain) which is connected to αNTD (Amino terminal domain) by
flexible linker.

• i. closed complex:

• Binding of RNA polymerase holoenzyme to the promotor sequence form closed comolex
RNA POLYMERASE
Mediates transcription of RNA from a double-stranded DNA template

Does not require primer

Transcription is always 5’ --> 3’ direction, with each base addition

using reaction RNAn + NTP --> RNAn+1 + PPi

Transcription inititation mediated by proteins that bind to specific

promoter sequence elements. The promoter binding proteins
recruit RNA polymerase to the initiation site.

RNA polymerases show evolutionary conservation between prokaryotes

and eukaryotes, accounting for many similarities in transcription
mechanism

But there are substantial differences in transcription initiation, termination,

and post-transcriptional RNA processing between pro- and eukaryotes
 PROKARYOTIC TRANSCRIPTION INITIATION

Prokaryotic promoters have elements near position -10 and -35 that bind
to the s initiation factor.
Promoter elements have been physically mapped by DNA footprinting.

Footprinting Technique
ELONGATION PROCEEDS IN A MOVING TRANSCRIPTION BUBBLE
 TRANSCRIPTION - TRANSLATION COUPLING IN PROKARYOTES

Prokaryotic mRNA does not require processing nor cell compartment

trafficking to become competent for translation.

Therefore, transcription and translation are coupled. In prokaryotes,

gene regulation is almost exclusively at level of transcription
initiation, and not later transcription or translation processes.
PROKARYOTIC TRANSCRIPTION TERMINATES BY RNA MECHANISM

r (rho) - independent
transcription termination
signal motif

r protein is recruited to RNA polymerase during elongation

to allow transcription termination at other signal motifs
 Termination
here are two mechanism of termination.
i. Rho independent:
•In this mechanism, transcription is terminated due to specific sequence in terminator DNA.
•The terminator DNA contains invert repeat which cause complimentary pairing as transcript RNA form hair pin structure.
•This invert repeat is followed by larger number of TTTTTTTT(~8 bp) on template DNA. The uracil appear in RNA. The load of hair pin structure is not tolerated by A=U base pair so
the RNA get separated from RNA-DNA heteroduplex.

ii. Rho dependent:

•In this mechanism, transcription is terminated by rho (ρ) protein.
•It is ring shaped single strand binding ATpase protein.
•The rho protein bind the single stranded RNA as it exit from polymerase enzyme complex  and hydrolyse the RNA from enzyme complex.
•The rho protein does not bind to those RNA whose protein is being translated. Rather it bind to RNA after translation.
•In bacteria transcription and translation occur simultaneously so the rho protein bind the RNA after translation has completed but transcription is still ON.
Transcription Regulation
Basal vs. activated transcription for
mRNA genes
A. General transcription factor (TF) vs. promoter-specific
1. general TFs are required by all mRNA genes
a. an absolute requirement
b. transcription can occur alone with these factors and by definition
the basal level of transcription
2. promoter-specific TFs are different for each gene
3. the promoter-specific TFs are required for maximal level of transcription
or for activated transcription (induction)
Prokaryotic cells turn genes on and off by controlling
transcription.

• A promotor is a DNA segment that allows a gene to be transcribed.

• An operator is a part of DNA that turns a gene “on” or ”off.”
• An operon includes a promoter, an operator, and one or more
structural genes that code for all the proteins needed to do a job.
• Operons are most common in prokaryotes.
• The lac operon was one of the first examples of gene regulation to be
discovered.
• The lac operon has three genes that code for enzymes that break down
lactose.
Lac Operon
• The lac operon acts like a switch.
– The lac operon is “off” when lactose is not present.
– The lac operon is “on” when lactose is present.
Eukaryotes regulate gene expression at many points.
• Different sets of genes are expressed in different types of
cells.
• Transcription is controlled by regulatory DNA sequences
and protein transcription factors.
• Transcription is controlled by regulatory DNA sequences
and protein transcription factors.
– Most eukaryotes have a TATA box promoter.
– Enhancers and silencers speed up or slow down the rate
of transcription.
– Each gene has a unique combination of regulatory
sequences.
• RNA processing is also an important part of gene regulation
in eukaryotes.
• mRNA processing includes three major steps.
 Spliced Gene Transcripts
• DNA sequence specifying a protein is broken into
segments (exons) scattered among longer noncoding
segments (introns).

• Initially, primary RNA transcript is produced for the entire

gene.
• Small nuclear ribonuclearproteins (snRNPs) associate with
proteins to form spliceosomes.
• Excise introns and splice exons to form mature mRNA.
RNA Splicing
• During RNA processing, intron sequences are cut out of primary
transcript before it is used in polypeptide synthesis.

• remaining exon sequences are spliced together to form final processed mRNA
Eukaryotic Genes are Fragmented
• mRNA processing includes three major steps.
– Introns are removed and exons are spliced together.
– A cap is added.
– A tail is added.