EXPRESSION OF GENETIC MATERIAL: FROM

TRANSCRIPTION TO TRANSLATION

SRESHTI BAGATI

Ph.D. Biotechnology

J-12-D-02-biot

CONTENT:

• 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:

Enzymes Location Product Sensitivity to α- amanitin

RNA Pol I Nucleolus rRNA Not sensitive

RNA Pol II Nucleoplasm mRNA Sensitive

RNA Pol III Nucleoplasm tRNA Sensitive in animals

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.

GENERIC PROMOTER:

• 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.

•Binds at the TATA box.

•Permits the association of TF II B and TF II A.

•Contains TBP and TAF’s.

TF II D

•Forms the DB complex.

•Helps the RNA polymerase II associates to the promoter site.

TF II B

•Accompanies RNA polymerase II to the promoter.

•Forms the transcription complex.

•Accelerates RNA chain growth uniformly.

TF II F

• 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 the obstructions.

• 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:

TERMINATIONIndicates the end of transcription and the release of the RNA

product.

ELONGATION

Involves the elongation of the growing RNA chain.INITIATION

Involves the formation of the promoter-polymerase complex.

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.

Source:www2.oakland.edu Source:www2.oakland.edu

2. Formation of open complex:

• The closed complex undergoes a transition.

• The DNA strands separate over a distance of some 14bp around the start site.

Source:www2.oakland.edu

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.

Source:www2.oakland.edu

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 transcription.

• TF II F accompanies the RNA polymerase to the promoter to form the transcription cycle.

• TF II E, TF II H (mediates promoter melting), TF II J helps in the initiation process.

Fig: Initiation of transcription in Eukaryotes

B. ELONGATION PHASE:

• The transition from initiation to elongation involves the shedding off of the initiation factors.

• 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.

Fig: Elongation phase in transcription.

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 :

I. 5’ untranslated region (5’ UTR)• varies in length.

II. The coding sequence• • specifies the amino acid sequence of the protein that will be produced

during translation.• • 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

the mRNA.

Fig. Structure of eukaryotic mRNA.

• PROCESSING OF RNA :

• In eukaryotes, the nascent RNA is called primary transcript-RNA.

• The processing steps are:

– capping

–polyadenylation

– RNA splicing

• The factors hSPT5 and TAT- SF1 help in the processing of the synthesized RNA strand.

1. 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 the5’end of a eukaryoticmRNA.

Source: www.wikipedia.org

2. POLYADENYLATION:

• 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.

Source: www.wikipedia.org

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

Source: www.molbiol4masters.masters.grkraj.org

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 SynthesisAngela N. Koehler, Alykhan F. Shamji, and Stuart L. Schreiber

Method: • 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 portion).

• 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 a control.

• This reveals that the above compound binds to GST portion of the Hap3p-GST fusion protein.

• 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 one).

• This indicates that compound 1 (Haptamide A) is a reversible inhibitor of Hap3p mediated transcription

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.

CONCLUSION:

• 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 DNA-protein interactions.

• DOS based search for inhibitors of such factors can be of therapeutic importance.

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:

1. mRNA’s

2. tRNA’s

3. Aminoacyl tRNA synthetases

4. Ribosome.

5. Translational factors.

TRANSLATIONAL FACTORS: are isolated from reticulocytes (an eukaryotic cell).

FACTOR FUNCTION

eIF3 Binding mRNA.

eIF4F Binding mRNA 5’end; unbinding

eIF1 Assists mRNA Binding

eIF4B Assists mRNA binding and unbinding

eIF4A Assists mRNA Binding

eIF6 Prevents 40S-60S joining

eIF5 Releasing eIF2 and eIF3

eIF4C Binding 60S sub unit

eIF2 Binding Met-tRNA, Binds to GTP, Recycling factor

eIF4D Unknown

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: A, P and E sites of Ribosome

Fig: Yeast 80S Ribosome Bound to the Sec61 1. Protein Complex Density corresponding to Sec61,

red; 2. P-site-bound peptidyl tRNA, green, 3. 40S proteins, aqua; 4. small subunit (18S) rRNA, yellow; 5. 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,

Source: en.wikipedia.org

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 DTT).

• 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.

RESULTS:• 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.

K

• 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 ribosome.

• 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 apart.

• 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).

CONCLUSION:

• 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 ternary complex.

•The ternary complex binds to the 40S ribosomal subunit.

• 43 initiation complex is formed.

• mRNA binds to this complex(eIF-3, eIF-4A, eIF-4B).

•  Initiation Complex travels till it reaches AUG( Scanning).

•60 S subunit binds to this complex (eIF-5).

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 enzyme).

•EF2 causes the translocation of newly formed P-tRNA and its codon from A to P site.

•Elongation continues…….

3.TERMINATION PHASE:

•Occurs when the termination codon occupies the A site.

•The release factor eRF1 leads to termination.

•It causes the dissociation of the two sub units.

•GTP is cleaved once the stop codon is reached , eRF1 is released.

INHIBITION OF TRANSLATION:

• Antibiotics :

1. kirromycin

2. Cyclohexamide.

• Protein toxins:

1. Ricin

2. α Sarcin

3. Diptheria

RNA 2008 14: 1290-1296 Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex motif.Amit Arora, Mariola Dutkiewicz, Vinod Scaria.

• The unusual secondary structures (formed from Guanine rich sequences) comprising of four Hoogsteen-paired coplanar guanines, known as G-quadruplexes (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 mRNA.

• 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.

Principle:

1. psiCHECK-2 vector from Promega was used as it allow simultaneous expression of Renilla and firefly luciferase from a single plasmid.

2. 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 (psiCHECK-2, GQ27, or GQ27m) were transfected into

HeLa cells, and 24 h after transfection, cells were harvested for dual luciferase assays.

MATERIALS AND METHODS:

1. OLIGONUCLEOTIDES:

The following RNA oligonucleotides for spectroscopic studies was purchasedfrom Chemgenes Corp.:

• Zic-1 5’-GGGUGGGGGGGGCGGGGGAGGCCGGGG-3’• Mutated Zic-1 5’-GAGUGAGGAGGACGAGAGAGGCCGAGG-3’

2. DNA oligonucleotides for PCR and cloning were purchased from TIB MOLBIOL.

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.

Black Bars: Protein activityGrey bars: relative mRNA levels.

RESULTS:• 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.

Summary:

• 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 charged t-RNA.

• Peptidyl transferase enzyme plays a key role in the formation of peptide bond.

• 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.

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