Expression of genetic material : From Transcription to Translation
EXPRESSION OF GENETIC
• Central Dogma Principle
• Transcription in Eukaryotes
• Post-Translational Modifications
• Inhibition of Transcription.
• Translation in Eukaryotes.
• Post Translational Modifications
• Inhibition of Translation
EXPRESSION OF GENETIC MATERIAL:
DNA stores all the genetic information in a stable form.
The expression of the genetic material requires its flow from DNA to RNA
and then to proteins.
This flow of information from DNA to proteins is known as Central Dogma
(Francis Crick ,1958).
TRANSCRIPTION IN EUKARYOTES:
Is defined as the process of synthesis of RNA from DNA.
The synthesized RNA represents the sense (coding) strand of DNA i.e., they
are identical in sequence.
Eukaryotic RNA Polymerase is of three types:
RNA Pol I
RNA Pol II
RNA Pol III
PROMOTERS OF RNA POLYMERASE II:
Are also called as cis-acting elements.
Promoter for RNA polymerase II consists of three regions, centered at sites
lying between -25bp and -100bp.
CAAT and GC box determine the efficiency of transcription.
A promoter sequence which is responsible for the constitutive expression of
the common genes in all cells, called as the house keeping genes.
A generic promoter is unable to bring about a regulated expression i.e. tissue
specific expression of genes (luxury genes).
TRANSCRIPTION FACTORS (TF’s):
are also known as trans –acting elements.
TF II D
• Binds at the TATA box.
• Permits the association of TF II B and TF II A.
• Contains TBP and TAF’s.
TF II B
• Forms the DB complex.
• Helps the RNA polymerase II associates to the promoter
TF II F
• Accompanies RNA polymerase II to the promoter.
• Forms the transcription complex.
• Accelerates RNA chain growth uniformly.
Other transcription factors are:
TF II E (helps the RNA polymerase II in leaving the promoter)
TF II H ( help in elongation)
TF II J (function unknown)
All these factors help in the initiation process.
The factor TF II S helps in the elongation of the RNA chain by removing
This factor acts by first causing the hydrolytic cleavage at the 3’ end of the
RNA chain and later leads to the forward movement of RNA polymerase.
MECHANISM OF TRANSCRIPTION:
For the transcription of a gene, RNA Polymerase proceeds through a series
of well defined steps. These are:
Involves the formation of the promoter-polymerase complex.
Involves the elongation of the growing RNA chain.
Indicates the end of transcription and the release of the RNA
A. INITIATION OF TRANSCRIPTION:
Is the first phase of the transcription cycle.
Involves three steps:
1. Formation of closed complex:
• initial binding of polymerase to the promoter.
• DNA remains double stranded, enzyme is bound to one face of the helix.
2. Formation of open complex:
The closed complex undergoes a transition.
The DNA strands separate over a distance of some 14bp around the start
3.FORMATION OF STABLE TERNARY COMPLEX:
The stable ternary complex formed contains the polymerase enzyme, DNA
and the RNA.
This marks the onset of elongation phase.
This is known as the pre-initiation complex.
ASSEMBLY OF ACTIVE TRANSCRIPTION COMPLEX:
TF II D binds to the TATA box and initiates transcription
The TATA box of DNA binds to the concave surface of TBP, a component
of TF II D.
TF II(TF II A and TF II B) guides RNA Polymerase II to the start site of
TF II F accompanies the RNA polymerase to the promoter to form the
TF II E, TF II H (mediates promoter melting), TF II J helps in the initiation
Fig: Initiation of transcription in Eukaryotes
B. ELONGATION PHASE:
The transition from initiation to elongation involves the shedding off of the
Elongation factors like ( TF II S and hSPT5) stimulate the elongation.
This exchange of factors is favoured by the phosphorylation of the CTD.
TF II S also shows proof reading activity.
C. TERMINATION OF TRANSCRIPTION:
In eukaryotes the termination is closely linked to an RNA processing event
called the 3’ polyadenylation.
The CTD tail of polymerase carries two protein complexes as it reaches the
end of a gene:
1. CPSF (cleavage and polyadenylation specificity factor)
2. CstF (cleavage stimulation factor)
These factors first cleave the RNA and then lead to polyadenylation.
STRUCTURE OF EUKARYOTIC mRNA:
Eukaryotic mRNAs have three main parts :
5’ untranslated region (5’ UTR)
varies in length.
II. The coding sequence
• • specifies the amino acid sequence of the protein that will be produced
• • It varies in length according to the size of the protein that it encodes.
III. 3’ untranslated region (3’ UTR)
• • also varies in length and contains information influencing the stability of
PROCESSING OF RNA :
In eukaryotes, the nascent RNA is called primary transcript-RNA.
• The processing steps are:
– RNA splicing
The factors hSPT5 and TAT- SF1 help in the processing of the synthesized
CAPPING OF THE 5’ END:
Involves the addition of a modified guanine (methylated guanine) base at
the 5’end of the RNA.
The 5’end of the RNA has a triphosphate moiety.
A guanine is added to the 5’end of the RNA. The 5’cap is added by an
unusual 5’-5’ linkage.
The reaction is catalyzed by Guanyl transferase.
Cap structure at the
5’end of a eukaryotic
• nascent RNA is cleaved downstream from the AAUAAA conserved sequence.
– By ribonuclease.
• The enzyme poly(A) polymerase adds adenine ribonucleotides
– up to 200 bases long at the 3’ end of the RNA.
• The poly(A) tail
– enhances the stability of eukaryotic mRNA and
– regulates its transport to the cytoplasmic compartment.
3. RNA SPLICING:
(RNA is called hnRNA - Heteronuclear RNA before splicing occurs).
• Splicing is:
– The mechanism by which introns are removed.
• Introns are intervening sequences - not expressed in proteins
• Exons are retained in the mature mRNA molecules.
– expressing sequences
• Exon and intron lengths and numbers vary in various genes.
MECHANISM OF SPLICING:
Involves the formation of spliceosome and transesterification reaction.
• Spliceosome has 4 different small nuclear ribonucleoprotein particles
assembled on mRNA.
1. snRNP U1 (binds at the left splice site)
2. snRNP U2 (binds at the right splice site)
3. snRNP U5 (binds at right,3’ splicing site)
4. snRNP U4/U6 (forms complex with snRNP U5)
The intron degrades, the two exons are ligated.
MECHANISM OF SPLICING.
INHIBITION OF TRANSCRIPTION:
• α- amanitin acts as the inhibitor of
RNA polymerase II, the key enzyme in
synthesis of mRNA.
α- amanitin is produced by the poisonous mushroom Amanita phalloides
(death cup or the destroying angel).
MODE OF ACTION
Dr. Bushnell et al., gave the crystal structure of α- amanitin .
Interacts with the bridge helix in the RNA Polymerase II.
Fig: Ribbon diagram of Saccharomyces cerevisiae, RNA Polymerase II in
complex with α- amanitin (red). Active site Magnesium ion visible in pink
near center (Bushnell et al.,)
Picture Source: en.wikipedia.org
Journal of American Chemical Society (2003).125
Discovery of an Inhibitor of a Transcription Factor Using Small Molecule
Microarrays and Diversity-Oriented Synthesis
Angela N. Koehler, Alykhan F. Shamji, and Stuart L. Schreiber
• A small molecule microarray based screen (identifies the DOS derived small
molecules, which modulate the transcriptional activity of Hap3p( a subunit of
Hap2/3/4/5p complex) was made.
Such small microarrays containing 12396 compounds (from 3 different DOS
pathways) were prepared in a one bead-one stock solution format.
These were then printed on to glass microscope slides.
3 micro arrays were probed with purified Hap3p-GST fusion protein (Binding
was detected using a Cy5-labelled anti GST antibody against the GST
Two reproducible positives 1 and 2 (from a library of dihydropyrancarboxamides)
were revealed on the screen.
Compound 2 also appeared as positive when the library was screened with GST as
This reveals that the above compound binds to GST portion of the Hap3p-GST
Compound 1 (haptamide A, inhibits the expression of reporter) binds to Hap3p
and modulates endogenous Hap3p function in cells (tested using GDH1-LacZ
reporter gene assay).
Negative control 2 did not affect the expression of the reporter.
Cells which were treated with haptamide A (60 min) were washed in fresh
medium (60 min), the expression levels in cells approached to normal (untreated
This indicates that compound 1 (Haptamide A) is a reversible inhibitor of Hap3p
Dose-response of 1 in a reporter gene assay.
BY4741 cells or BY4741 hap3 cells (*)
expressing a GDH1-LacZ reporter were
treated with 1 or 2.
Certain transcription factors become over active in case of cancers.
Such factors promote metastasis and tumour growth.
Such factors can be inhibited by disrupting the protein-protein or the
DOS based search for inhibitors of such factors can be of therapeutic
TRANSLATION IN EUKARYOTES:
The translation of gene transcripts(mRNA) into proteins is the final step of
the expression of the genome (Hinnebusch, 2000).
High throughput analysis of (ribosome+mRNA), transcriptome of
Saccharomyces cerevisiae opened the way for investigations of translational
control at the genome-wide level (Arava et al., 2003; MacKay et al., 2004
The translational machinery includes four primary components:
Aminoacyl tRNA synthetases
TRANSLATIONAL FACTORS: are isolated from reticulocytes (an eukaryotic
Binding mRNA 5’end; unbinding
Assists mRNA Binding
Assists mRNA binding and unbinding
Assists mRNA Binding
Prevents 40S-60S joining
Releasing eIF2 and eIF3
Binding 60S sub unit
Binding Met-tRNA, Binds to GTP, Recycling factor
STRUCTURE OF RIBOSOME:
Is a spheroid structure of 150 to 250A in diameter.
• Eukaryotic ribosome is 80S and has two subunits:
1. Larger subunit (60S)
2. Smaller subunit (40S): is responsible for binding and decoding messenger
RNA (ensures correct binding).
The 40S subunit (18S rRNA and 32 ribosomal proteins) in S.cerevisiae is
divided into head, body and platform.
The 60S subunit (28S, 5.8S, 5S rRNA) and contains the expansion segment
as well as the peptidyl transferase active site.
Fig: Yeast 80S Ribosome Bound to the Sec61
Protein Complex Density corresponding
2. P-site-bound peptidyl tRNA, green,
3. 40S proteins, aqua;
4. small subunit (18S) rRNA, yellow;
60S proteins orange;
6. large subunit (25S/5.8S/5S) rRNAs, blue.
Structure and Function of the Eukaryotic Ribosome: The
Next Frontier Jennifer A. Doudna (et.al.)
Cell, Vol. 109, 153–156, April 19, 2002,
Fig: A, P and E sites of Ribosome
Nucleic Acids Research, (1998) 26 :2 655–661
Three-Dimensional Structure of the Yeast of the Yeast Ribosome.
Adriana Verschoor, Jonathan R. Warner, Suman Srivastava1, Robert A. Grassucci
and Joachim Frank.
Materials and Methods:
• Ribosomes were isolated from S.cerevisiae strain W303.
Cells were collected by centrifugation, washed in water and resuspended in
TMN (50mM Tris-acetate, pH 7.4, 50mM NH4 Cl, 12mM MgCl2, 1mM
Cells were broken down by vigorous agitation with glass beads. Ribosomes
were separated from other soluble proteins.
Grids were prepared for Cryo microscopy using the standardized methods
(Wagenknecht and Dubochet et al.)
Micrographs were recorded using Philips EM420 at a magnification
of38000X; the resulting pixel size was 5.26 A
12 micrographs at 0 , 35 and 50 were analyzed. Analysis was of 7470
projections with a projection matching scheme (Wheat germ structure was
taken as an initial reference).
Finally, all data sets were pooled and the SIRT back projection
algorithm was iterated until the results stabilized.
• The 80S ribosome from S.cerevisiae has been reconstructed from 7470
individual ribosome images.
The yeast ribosome is a bipartite structure, varying from roughly ellipsoidal
to somewhat elongated.
In the above figure the 2 sub units are seen side by side,40S (left) and 60S
(right), with their “heads” at the top.
The height of the ribosome is ~254 Å, the width ~278 Å and the
thickness ~267 Å which is 11-14% lesser than that of wheat germ
The 2 subunits of the yeast ribosome are clearly separated, can be seen side
by side (E and J).
K depicts the presence of a plane that can be interposed to cut the sub units
The larger bridge links the mid portions of the sub units (flattish surface of
60S and platform interface of 40S. (A and J).
The smaller bridge links the bases of the subunits ( E and J).
The principal difference between the 80S ribosome of yeast and wheat
germ is :
1. The yeast ribosome is more compact in height and width.
2. Yeast ribosome is more globular, and is more thick.
3. The resolution into two separate sub units is superior for yeast.
There are a number of yeast ribosomal proteins which are not essential for
life but are needed for optimal assembly and the function of ribosome.
Presence of some mutant ribosomal proteins which have a substantial effect
on the accuracy of the ribosomes translational efficiency has been detected.
STRUCTURE OF tRNA:
Holley sequenced the first tRNA in 1965 and suggested the presence of
several secondary structures (Holley et al., 1965).
The most accepted model for the structure of tRNA is Clover leaf Model.
The three dimensional structural model of tRNA was given by Levitt
(Levitt, 1969) which was proved wrong.
the first correct structure of tRNA chain in crystals was done in yeast
tRNAPhe in 1974 (Suddath et al., 1974).
L shaped 3D structure of tRNA
STEPS INVOLVED IN PROTEIN SYNTHEIS:
1. INITIATION PHASE :
Amino acid activation:
1. Amino acid + ATP
Aminoacyl-AMP + PPi
2. Aminoacyl-AMP + tRNA
aminoacyl-tRNA + AMP
3. Amino acid+ ATP+ tRNA
aminoacyl-tRNA + AMP + Ppi.
Formation of ternary complex.
Binding of ternary complex to 40S subunit.
Binding of mRNA; Scanning of Initiation Codon (AUG).
Formation of 80S ribosomal unit.
•Initiation factor eIF-2 is a GTP-binding
protein that specifically recognizes
initiator tRNA (Met-tRNAMet) forming a
•The ternary complex binds to the 40S
• 43 initiation complex is formed.
• mRNA binds to this complex(eIF-3, eIF4A, eIF-4B).
• Initiation Complex travels till it reaches
•60 S subunit binds to this complex (eIF5).
2. ELONGATION PHASE:
•Addition of second codon of mRNA
to AA in the tRNA (EF1 and GTP
hydrolysis is needed).
•Peptide bond is formed between the
amino group of aa-tRNA and carboxyl
group of p-tRNA (peptidyl transferase
•EF2 causes the translocation of newly
formed P-tRNA and its codon from A
to P site.
•Occurs when the termination
codon occupies the A site.
•The release factor eRF1 leads to
•It causes the dissociation of the
two sub units.
•GTP is cleaved once the stop
codon is reached , eRF1 is
RNA 2008 14: 1290-1296
Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex
Amit Arora, Mariola Dutkiewicz, Vinod Scaria.
The unusual secondary structures (formed from Guanine rich sequences)
comprising of four Hoogsteen-paired coplanar guanines, known as Gquadruplexes (GQ) , Gellert et al. 1962.
Small molecules that bind and stabilize these RNA-GQs suppress the gene
expression of certain oncogenes, hence can be used in therapeutics ( Rangan
et al. 2001; Siddiqui-Jain et al. 2002).
This study was confined to the GQ motifs located in the 5’- UTRs of
For this the mRNA of the zinc-finger protein Zic-1 was chosen.
Aim: Investigation of RNA-GQ motif on
translation in living Eukaryotic cells.
1. psiCHECK-2 vector from Promega was
used as it allow simultaneous expression
of Renilla and firefly luciferase from a
For the evaluation of the influence of
Zic1-RNA GQ on translation:
A 27bp long DNA sequence encoding the
GQ motif was cloned upstream of the
Renilla luciferase. The plamsid so
formed is known as GQ27.
The three vector constructs (psiCHECK2, GQ27, or GQ27m) were transfected into
HeLa cells, and 24 h after transfection, cells
were harvested for dual luciferase assays.
MATERIALS AND METHODS:
The following RNA oligonucleotides for spectroscopic studies was
purchasedfrom Chemgenes Corp.:
• Mutated Zic-1 5’-GAGUGAGGAGGACGAGAGAGGCCGAGG-3’
2. DNA oligonucleotides for PCR and cloning were purchased from TIB
3. GQ27 plasmid was constructed.
4. HeLa cells were cultured and the dual luciferase assay (using
spectrophotometer) was carried out.
5. Total RNA isolation and RT-PCR studies were carried out.
• Original Renilla and firefly luciferase ratio was found to be around 30.
• Insertion of Zic1-RNA GQ27 reduces protein synthesis by 80%.
• Mutated GQ27 did not effect the translation.
Black Bars: Protein activity
Grey bars: relative mRNA levels.
Gene expression is the process by which the information in the DNA gets
converted into RNA and then proteins.
The first step (Transcription) is catalyzed by RNA Pol II and the mRNA is
synthesized in the 5’-3’ direction.
The second step (Translation) involves the use of translational factors and
Peptidyl transferase enzyme plays a key role in the formation of peptide
The products of both the processes undergo certain modifications before
they move out of their site of synthesis. i.e. mRNA undergoes
capping, polyadenylation and splicing where as proteins undergo folding
and localization before they start their action.
Asano, K., Kinzy, T.G., Merrick, W.C., and Hershey, J.W.B. 1997.
Conservation and diversity of eukaryotic translation initiation factor eIF3. J.
Biol. Chem. 272: 1101–1109.
Grosjean H and Benne R .1998 .Modification and Editing of RNA.
Washington, DC: American Society for Microbiology.
Benjamin Lewin,2000. Genes VII , pp.617-685. Oxford University Press
Inc., New York.
Berg, Tymoczko, Stryer,2002.Biochemistry (Fifth edition),pp.129-137.W.H
.Freeman and Company, New York.
Watson,Baker,Bell,Gann,2003.Molecular Biology of the Gene(Fifth
Koehler,Shamji,Schreiber,2003.Discovery of an inhibitor of a Transcription
Factor Using Small Molecule Microarrays and Diversity Oriented
Synthesis, American Chemical Society.125: 8420-8421.
Verma and Aggarwal,2005. Cell Biology, Molecular Biology, Evolution and
Ecology, pp. 45-90. S.Chand & Company , India.
Victoria G. Kolupaeva, Anett Unbehaun, Ivan B. Lomakin, 2005. Binding of
eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in
ribosomal association and anti-association. RNA 11: 470-486.
Julie E. Takacs, Timothy B. Neary, Nicholas T. Ingolia, 2011 Identification of
compounds that decrease the fidelity of start codon recognition by the
eukaryotic translational machinery. RNA 17: 439-452