Transcription & Translation

Science Of Living System

Overview of Nucleic Acids ,Transcription, Translation and Recombinant DNA

Technology

BY

School of Medical Science and Technology,

Indian Institute of Technology Kharagpur

Copyrights@ Prof.A.K.Ghosh

Eukaryotic cells with a nucleus

• Nucleus• Mitochondria• Chloroplast• Ribosomes• RER• SER• Golgi body• Cytoplasm• Vacuoles

Prokaryotic cellswithout a nucleus

• Cytoplasm• Ribosomes• Nuclear Zone• DNA• Plasmid• Cell Membrane• Mesosome• Cell Wall• Capsule (or slime layer)• Flagellum


Macromolecules

Protein

Nucleic acids

Olygosaccharides

Lipids

Complex macromoleculesCopyrights@ Prof.A.K.Ghosh

Cell Nucleus, compartmentalized DNA activityNuclear pores allow communicationNuclear lamina and cytoskeleton mechanically support the nucleus


Chromosomes at interphase and M phase


Human ChromosomeComplex of DNA and protein is called chromatin44 homologous chromosomes and 2 sex chromosomesComplementary DNA with different DyesThe arrangement of the full chromosome set is called karyotype


Banding Pattern of human chromosomesGiemsa StainingGreen line regions: centromeres

Encoding ribosome


Conservation between human and mouse genomesUsually important genes are encoded by conserved regionsNote: Human chromosome 1 and mouse chromosome 4

human mouse

centromere


DNA Double Helix10.4 nucleutides/turn; 3.4 nm between nucleutides


DNA Molecules are highly condensed in chromosomesNucleosomes of interphase under electron microscopeNucleosome: basic level of chromosome/chromatin organization Chromatin: protein-DNA complexHistone: DNA binding proteinA: diameter 30 nm; B: further unfolding, beads on a string conformation


Chromatin PackingCondensin plays important roles


Nucleosome StructuresHistone octamer2 H2A2 H2B2 H32 H4


The function of Histone tails


The bending of DNA in a nucleosome1. Flexibility of DNAs: A-T riched minor groove inside and G-C riched groove outside2. DNA bound protein can also help


Irregularities in the 30-nm fiberFlexible linker, DNA binding proteinsStructural modulators: H1 histone, ATP-driven Chromatin remodeling machine, covalent modification of histone tails


• A gene is a nucleotide sequene in a DNA molecule that act as a functional unit for protein production, RNA synthesis.

• Introns and Exons• Chromosome: single long DNA contains a linear array of

many genes. • Human genome contains 2.3x109 DNA nucleotide pairs,

with 22 different autosomes and 2 sex chromosomes.• Chromosomal DNA: replication origins, telomeres,

centromeres• Histones form the protein core for DNA wrapping• Nucleosome: repeating array of DNA-protein particles• Modification of Chromatin and nucleosomes: histone H1,

ATP-driven chromatin remodeling complexes, and enzymatically catalyzed covalent modification of the N-terminal tails of Histones

Chromosomal DNA and its Packaging



DNA to ProteinGenome: the complete set of information in an organism’s DNATotal length of DNA about 2 meters long in a human cell, encoding about 30000 proteins


Structural Organization of the Core Histones


Nucleic acids

• Deoxyribonucleic acid (a polymer of deoxyribonucleotides)

• Ribonucleic acid (a polymer of ribonucleotides)

A nucleotide is made up of Sugar, Nitrogenous base and phosphate


DNA RNA


DNA RNA


Nucleotide tri phosphate





Organization of DNA molecule


DNA consists of two strands running anti-parallel and forming double hellical structure


DNA Structure

• Conclusion-DNA is a helical structure with distinctive regularities, 0.34 nm & 3.4 nm.


RNA

• RNA is a polymer ribonucleotides that contains ribose rather than deoxyribose sugars. The normal base composition is made up of guanine, adenine, cytosine, and uracil

• RNA is found in nucleus and cytoplasm

• Types of RNA : Messenger RNA (mRNA) Ribosomal RNA ( rRNA)

Transfer RNA (tRNA)

Consult: http://www.biology.arizona.edu/biochemistry/activities/DNA/04q.htmlCopyrights@ Prof.A.K.Ghosh

RNA StructureMore commonly, RNA is single stranded and can form complex and unusual shapes.


DNA vs. RNA

• DNA • Double Helix• Deoxyribose sugar• Adenine pairs with

Thymine (A-T)• Stays in nucleus

• RNA• Single strand• Ribose sugar• Uracil replaces Thymine• Leaves nucleus to do

the work






James D. Watson & Francis H. Crick

• In 1953 presented the double helix model of DNA• Two primary sources of information:

– 1. Chargaff Rule: #A#T and #G#C. “A strange but possibly meaningless phenomenon”.

– 2. X-ray diffraction studies of Rosalind Franklin & Maurice H. F. Wilkins


1962: Nobel Prize in Physiology and Medicine

James D.Watson

Francis H.Crick

Maurice H. F.Wilkins

What about?Rosalind Franklin

Watson, J.D. and F.H. Crick, “Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic Acids”. Nature 171 (1953), p. 738.






Palindromic sequence can be read in the same way in either direction - DNA is palindromic if it is equal to its complementary sequence read back-word ( TTAGCAC is

palindromic to CACGATT). It may form the hairpin.Mirror repeat : A DNA mirror repeat is a sequence segment delimited on the basis of

its containing centre of symmetry on a single strand and identical terminal nucleotides.

Tandem Repeat : It occurs in a DNA when a pattern of two or more nucleotides are repeated in a sequence.



Accuracy of DNA Replication

• The chromosome of E. coli bacteria contains about 5 million bases pairs

– Capable of copying this DNA in less than an hour

• The 46 chromosomes of a human cell contain about 6 BILLION base pairs of DNA!!

– Printed one letter (A,C,T,G) at a time…would fill up over 900 volumes of Campbell.

– Takes a cell a few hours to copy this DNA

– With amazing accuracy – an average of 1 per billion nucleotides


Identical base sequences

5’

5’

3’

3’ 5’

5’3’

3’

Watson/Crick proposed mechanism of DNA replication Copyrights@ Prof.A.K.Ghosh

1955: Arthur Kornberg

Worked with E. coli. Discovered the mechanisms of DNA synthesis.

Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)

2. DNA template

3. DNA polymerase (Kornberg enzyme)

4. Mg 2+ (optimizes DNA polymerase activity)

1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)Copyrights@ Prof.A.K.Ghosh

Intermission


DNA Replication

• DNA must replicate during each cell division• 3 alternative models for DNA replication were hypothesized:

– Semiconservative replication

– Conservative replication

– Dispersive replication

ConservativeSemi-conservative Dispersive Copyrights@ Prof.A.K.Ghosh

DNA Replication is “Semi-conservative”

• Each 2-stranded daughter molecule is only half new

• One original strand was used as a template to make the new strand


DNA Replication• The copying of DNA is remarkable in its speed and accuracy

• Involves unwinding the double helix and synthesizing two new strands.

• More than a dozen enzymes and other proteins participate in DNA replication

• The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated


Origins of Replication• A eukaryotic chromosome may have hundreds or

even thousands of replication origins

Replication begins at specific siteswhere the two parental strandsseparate and form replicationbubbles.

The bubbles expand laterally, asDNA replication proceeds in bothdirections.

Eventually, the replicationbubbles fuse, and synthesis ofthe daughter strands iscomplete.

1

2

3

Origin of replication

Bubble

Parental (template) strand

Daughter (new) strand

Replication fork

Two daughter DNA molecules

In eukaryotes, DNA replication begins at many sites along the giantDNA molecule of each chromosome.

In this micrograph, three replicationbubbles are visible along the DNA ofa cultured Chinese hamster cell (TEM).

(b)(a)

0.25 µm


Mechanism of DNA Replication

• DNA replication is catalyzed by DNA polymerase III which needs an RNA primer

• DNA polymerase III cannot initiate the synthesis of a polynucleotide, they can only add nucleotides to the 3 end

• The initial nucleotide strand is an RNA primer

• RNA primase synthesizes primer on DNA strand

• DNA polymerase adds nucleotides to the 3’ end of the growing strand

DNA polymerase I degrades the RNA primer and replaces it with DNA

DNA polymerase III adds nucleotides to primer


Mechanism of DNA Replication• Nucleotides are added by complementary base pairing with the template strand• DNA always reads from 5’ end to 3’ end for transcription replication • During replication, new nucleotides are added to the free 3’ hydroxyl on the

growing strand• The nucleotides (deoxyribonucleoside triphosphates) are hydrolyzed as added,

releasing energy for DNA synthesis.

• The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

New strand5 end

Phosphate BaseSugar

Template strand3 end 5 end 3 end

5 end

3 end

5 end

3 end

Nucleosidetriphosphate

DNA polymerase

Pyrophosphate


The Mechanism of DNA Replication

• DNA synthesis on the leading strand is continuous

• Only one primer is needed for synthesis of the leading strand

• The lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5’-to-3’ direction

• Therefore, DNA synthesis on the lagging strand is discontinuous

• For synthesis of the lagging strand, each fragment (Okazaki) must be primed separately, then DNA fragments are sythesized and subsequently ligated together

Parental DNA

5

3

Leading strand

35

3

5

Okazakifragments

Lagging strand

DNA pol III

Templatestrand

Leading strand

Lagging strand

DNA ligase Templatestrand

Overall direction of replicationCopyrights@ Prof.A.K.Ghosh

Mechanism of DNA Replication• Many proteins assist in DNA replication

– DNA helicases unwind the double helix, the template strands are stabilized by other proteins

– Single-stranded DNA binding proteins make the template available

– RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added.

– DNA polymerase III extends the strand in the 5’-to-3’ direction

– DNA polymerase I degrades the RNA primer and replaces it with DNA

– DNA ligase joins the DNA fragments into a continuous daughter strand

5

3Parental DNA

3

5

Overall direction of replication

DNA pol III

Replication fork

Leadingstrand

DNA ligase

Primase

OVERVIEW

PrimerDNA pol III

DNA pol I

Laggingstrand

Laggingstrand

Leadingstrand

Leadingstrand

Laggingstrand

Origin of replication


Enzymes in DNA replication

Helicase unwinds parental double helix

Binding proteinsstabilize separatestrands

DNA polymerase III binds nucleotides to form new strands

Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone

Primase adds short primer to template strand

DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct basesCopyrights@ Prof.A.K.Ghosh

Binding proteins prevent single strands from rewinding.

Helicase protein binds to DNA sequences called origins and unwinds DNA strands.

5’

3’

5’

3’

Primase protein makes a short segment of RNA complementary to the DNA, a primer.

3’ 5’

5’ 3’

Replication


Overall directionof replication

5’ 3’

5’

3’

5’

3’

3’ 5’

DNA polymerase III enzyme adds DNA nucleotides to the RNA primer.

Replication


5’

5’

Overall directionof replication

5’

3’

5’

3’

3’

3’

DNA polymerase proofreads bases added and replaces incorrect nucleotides.

Replication


5’

5’ 3’

5’

3’

3’

5’

3’Overall directionof replication

Leading strand synthesis continues in a 5’ to 3’ direction.

Replication


3’ 5’ 5’

5’ 3’

5’

3’

3’

5’


Okazaki fragment


Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication


3’ 5’ 5’

5’ 3’

5’

3’

3’

5’


Okazaki fragment



Replication


5’ 5’

5’ 3’

5’

3’

3’

5’


3’



Okazaki fragment

Replication


5’

5’ 3’

5’

3’

3’

5’

3’

3’

5’ 5’ 3’



Replication


3’

5’

3’

5’

5’ 3’

5’

3’

3’

5’ 5’ 3’



Replication


5’

5’

3’ 3’

5’

3’

5’ 3’

5’

3’

3’

5’

Exonuclease activity of DNA polymerase I removes RNA primers.

Replication


Polymerase activity of DNA polymerase I fills the gaps.Ligase forms bonds between sugar-phosphate backbone.

3’

5’

3’

5’ 3’

5’

3’

3’

5’

Replication


Replication Fork Overview

5

3Parental DNA

3

5

Overall direction of replication

DNA pol III

Replication fork

Leadingstrand

DNA ligase

Primase

OVERVIEW

PrimerDNA pol III

DNA pol I

Laggingstrand

Laggingstrand

Leadingstrand

Leadingstrand

LaggingstrandOrigin of replication


3

Polymerase III

5’ 3

’

Leading strand

base pairs

5’

5’

3’

3’

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

Helicase +

Initiator Proteins

ATP

SSB Proteins

RNA Primer

primase

2Polymerase III

Lagging strand

Okazaki Fragments

1

RNA primer replaced by polymerase I& gap is sealed by ligase



Other Proteins That Assist DNA Replication• Helicase, topoisomerase, single-strand binding

protein are all proteins that assist DNA replication


Proofreading• Mistakes during the initial pairing of template

nucleotides and complementary nucleotides occur at a rate of one error per 100,000 base pairs.

• DNA polymerase proofreads each new nucleotide against the template nucleotide as soon as it is added and can correct errors

• If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis.

• Mismatched nucleotides that are missed by DNA polymerase or mutations that occur after DNA synthesis is completed can often be repaired


Proofreading and Repairing DNA

• DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides

• In mismatch repair of DNA, repair enzymes correct errors in base pairing

• In nucleotide excision DNA repair nucleases cut out and replace damaged stretches of DNA

Nuclease

DNApolymerase

DNAligase

A thymine dimerdistorts the DNA molecule.1

A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.

2

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

3

DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.

4


DNA repair


Replicating the Ends of DNA Molecules• The ends of eukaryotic chromosomal DNA get

shorter with each round of replication

End of parentalDNA strands

Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strand

Removal of primers andreplacement with DNAwhere a 3 end is available

Primer removed butcannot be replacedwith DNA becauseno 3 end available

for DNA polymerase

Second roundof replication

New leading strand

New lagging strand 5

Further roundsof replication

Shorter and shorterdaughter molecules

5

3

5

3

5

3

5

3

3


Replicating the Ends of DNA Molecules• The ends of eukaryotic chromosomal DNA get

shorter with each round of replication

End of parentalDNA strands

Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strand

Removal of primers andreplacement with DNAwhere a 3 end is available

Primer removed butcannot be replacedwith DNA becauseno 3 end available

for DNA polymerase

Second roundof replication

New leading strand

New lagging strand 5

Further roundsof replication

Shorter and shorterdaughter molecules

5

3

5

3

5

3

5

3

3


Telomeres• Eukaryotic chromosomal DNA

molecules have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules


Telomerases• If the chromosomes of germ cells

became shorter in every cell cycle essential genes would eventually be missing from the gametes they produce

• An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells


DNA polymerase catalyzed

nucleophilic attack of the 3’-OH on a

phospho-anhydride

** Since the 3’ –OH is changed to a –H in ddNTPs, it is unable to form a phosphodiester bond by nucleophilic attack on the phosphate, and it will cause a termination in the DNA chain

Mechanism of DNA polymerization

::

O

HO

HH

HH

PO

O-

O-

O

HO

HH

HH

PO O-

Base

Base

O

HOH

HH

HH

OBase

O

P-O O-

O-

5’

3’

O

HOH

HH

HH

OPO

O-

O

POP-O

O

O-

O

O-

Base

O

HO

HH

HH

PO

O-

O

O-

O

HO

HH

HH

PO O-

Base

Base

O

HO

HH

HH

OBase

PO O-

O

HOH

HH

HH

OBase

O

P-O

O-

5’

3’

OP-O

O-

O

OHP

O-

O


Polymerase Chain Polymerase Chain Reaction (PCR) and Its Reaction (PCR) and Its

ApplicationsApplications


What is PCR?What is PCR?

It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.


What is PCR? : What is PCR? : Why “Polymerase”?Why “Polymerase”?

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.


What is PCR? : What is PCR? : Why “Chain”?Why “Chain”?

It is called “chain” because the products of the first reaction become substrates of the following one, and so on.


What is PCR? : What is PCR? : The “Reaction” ComponentsThe “Reaction” Components

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequence to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction

5) Mg++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme Copyrights@ Prof.A.K.Ghosh

The ReactionThe Reaction

THERMOCYCLERPCR tube


Denature (heat to 95oC)

Lower temperature to 56oC Anneal with primers

Increase temperature to 72oC DNA polymerase +

dNTPs



Thanks


The general molecular formula of an amino acid is RCH(NH2)COOH

C

C

R

OO

NH

H

H

H

carboxylic acid group

amino group

Side chain:

‘R’ characterises the amino acid

Proteins are made up of one or more polypeptide. Each polypeptide is a chain of co-valently bonded amino acids

Proteins


Formation of the peptide bond

O

NH2

R1OH

O

NH2

R2OH

Two amino acid molecules; the nature of the R group (R1 and R2) determines the amino acid

O

NH2

R1OH

O

NH2

R2

OH

The molecules must be orientated so that the carboxylic acid group of one can react with the amine group of the other

O

NH2

R1NH

O

R2

OH

OH2

The peptide bond forms with the elimination of a water molecule; it is another example of a condensation reaction



S G

Y

A

V


The levels of protein structure


http://www.ncbi.nlm.nih.gov Copyrights@ Prof.A.K.Ghosh

Today




DNA

mRNA

Transcription

The Central Dogma of Molecular Biology

Cell

Polypeptide

(protein)

Translation Ribosome

This describes the flow of information from DNA into RNA (most commonly mRNA) through transcription (copying the same code from one molecule to another), and then expressing the code into a functional molecule by translation (converting from a nucleic acid code into an amino acid code).Copyrights@ Prof.A.K.Ghosh

• Gene: a segment of DNA containing the information for a single polypeptide chain or functional RNA (i.e. rRNA, tRNA, etc.)

Basic Gene Structure

transcription

5’ ACAU…AUG…UGA…AUGA 3’ RNA

Transcribed region

Regulatory elements

5’ ACAT…ATG…TGA…ATGC 3’3’ TGTA…TAC…ACT…TACT 5’

Promoter Terminator DNA

Gene

+1 Downstream (+n)Upstream (-n)

RNAP

• The site at which RNA polymerase begins transcription is numbered +1. - Downstream: direction in which a template DNA strand is transcribed - Upstream: denotes the opposite direction - Nucleotide positions in the DNA sequence downstream are indicated by (+) and those downstream (-). Which DNA strand is the template strand?


DNA RNA Protein

Gene Expression

transcription

Copying of information

• During transcription, the 4 base language of DNA is simply copied into the 4 base language of RNA.

translation

• During protein synthesis, the 4 base language of RNA is translated into the 20-amino acid language of proteins.


Eukaryotic cells with a nucleus

• Nucleus• Mitochondria• Chloroplast• Ribosomes• RER• SER• Golgi body• Cytoplasm• Vacuoles

Prokaryotic cellswithout a nucleus

• Cytoplasm• Ribosomes• Nuclear Zone• DNA• Plasmid• Cell Membrane• Mesosome• Cell Wall• Capsule (or slime layer)• Flagellum


TRANSCRIPTION AND TRANSLATION:Prokaryotic vs. Eukaryotic

SEPARATE COMPARTMENTSCOUPLED


7

Prokaryotic Gene StructurePromoter CDS Terminator

transcription

Genomic DNA

mRNA

protein

UTR UTR

translation

Promoter is a DNA sequence usually present upstream of coding regions where RNA polymerase binds to initiates transcription.

Gene is the structural and functional unit of heridity which carry genetic information from one generation to next. In molecular terms Gene is a part of chromosomes (DNA) which codes for functional RNA or protein

Gene transcription in prokaryotes

UTR (Untranslated sequences): 5’ UTR contains ribosome binding sites for protein synthesis; 3’UTR helps in stability of RNA

CDS: coding sequences for protein synthesis

Terminator: Sequence for ending RNA synthesisCopyrights@ Prof.A.K.Ghosh

13

Promoter

• Promoters sequences can vary tremendously.

• RNA polymerase recognizes hundreds of different promoters

5'

3'

3'

5'-50 -40 -30 -20 -10 1 10

start -10 region

T A T A A T A T A T T A

(Pribnow box)

-35 region

T T G A C A A A C T G T

Prokaryotic promoter

Consensus sequenceCopyrights@ Prof.A.K.Ghosh

RNA transcript is complementary to template strand and identical to coding strand

Requirement for transcription in prokaryotesGene or DNA to be transcribed, RNA polymerase, rNTPS

and cellular environment

Different genes are transcribed from different strands


Bacteria has one RNA polymerase to synthesize all three RNA: (mRNA, rRNA, tRNA)

RNA polymerase binds to promoter of a gene to initiate transcriptionCopyrights@ Prof.A.K.Ghosh

The Holoenzyme:Direction of transcription

Upstream Downstream

Prokaryotic RNA polymerase

-Core pol + 70 factor = RNA Pol Holoenzyme-70 factor recognizes and binds to the promoter region


Stages of Transcription

• Chain Initiation

• Chain Elongation

• Chain Termination


Figure 7-9 Essential Cell Biology (© Garland Science 2010)

Promoter and terminator sequences of a gene tell the RNA polymerase where to start and stop transcription


Figure 7-10 Essential Cell Biology (© Garland Science 2010)

Promoter and terminator sequences of a gene tell the RNA polymerase where to start and stop transcription


Bacterial Transcription Initiation

• Promoter recognition by RNA polymerase

• Formation of Transcription Bubble by separating DNA strands

• Bond creation between rNTPs to start RNA synthesis

• Escape of transcripton apparatus from promoter


How RNA Polymerase finds promoter and Initiates Transcription

•Core enzyme has the ability to synthesize RNA on a DNA template but cannot initiate transcription at proper site

•Core polymerase has general affinity for DNA and loosely binds at random sites in DNA without discriminating promoter and other sequences.

•Binding of sigma introduce a major changes in the polymerase and the holoenzyme drastically reduced ability to recognize loose binding sites, and the enzymes moves along the DNA by directly displaced by another sequences.

•When it reaches the promoter sequences, sigma factor recognize specifically -35 sequence and binds tightly.

•The holoenzyme occupies -40 to +20 regions of DNA and unwinds DNA (17 bp) from -10 regions and adds ribonucleotide (G or A) in the +1 site.

•After the synthesis of 6-9 nucleotides long RNA without movement of enzyme, sigma factor falls off from holoenzyme and the core enzyme enters the elongation process [promoter escape]


Finding and binding the promoter

Closed complex formation

RNAP bound -40 to +20

Open complex formation

RNAP unwinds from -10 to +2

Binding of 1st NTPRequires high purine [NTP]

Addition of next NTPsRequires lower purine [NTPs]

Dissociation of sigmaAfter RNA chain is 6-10 NTPs long

initiation


Transcription Process:Initiation Elongation Termination

“Closed Complex”

“Open complex”

(~14 bps)

(Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond.)


(primary transcript)

Transcription Process:Initiation Elongation Termination


A closer look at transcription termination: Intrinsic Terminators

G/C rich A rich•Terminator is usually a G/C-rich sequence followed by an A-rich sequence.

•An RNA:RNA stem loop forms (more stable due to G/C-rich in stem). This is followed by the formation of DNA:RNA hybrid via A-U pairing.

•Release of RNA chain since A-U base pairing is less stable and is easily dissociated.


Structure of Prokaryotic RNA Polymerase

Upstream DNA

Downstream DNA

-Prokaryotic RNA pol is composed of 5 subunits: 2, , ’ and (Core enzyme).Copyrights@ Prof.A.K.Ghosh

Transcription: RNA Synthesis

•One DNA strand acts as a template (read in the 3’5’), determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA strand.

•This polymerization reaction is catalyzed by RNA polymerase

•Polymerization involves a nucleophilic attack by the 3’ oxygen in the growing RNA chain on the phosphate if the next nucleotide to be added…resulting in a phosphodiester bond (energetically favorable).

•RNA synthesis: 5’3’Copyrights@ Prof.A.K.Ghosh

Subsequenthydrolysis ofPPi drives thereaction forward

RNA strand

OH

OH

DNA strand

RNA Synthesis is in the 5’ to 3’ Direction

RNA has polarity (5’ phosphate, 3’ hydroxyl)Copyrights@ Prof.A.K.Ghosh

RNA polymeraseelongation

זיווגי בסיסים12-כ "עין"ה

•During Elongation RNA polymerase unwinds DNA ahead of it, transcribe the region and rewinds the DNA at the back and RNA comes out of the complex.

•Transcription occurs in the Transcription•Bubble at the rate of 50 nt/sec.

•Elongation continues till Core enzymes reaches the terminator sequences.• Copyrights@ Prof.A.K.Ghosh

Transcription Termination

Transcription ends after a terminator is transcribed

• Two types of terminators in bacteria:

– Rho-dependent terminators

– Rho-independent terminators

7

Prokaryotic Gene StructurePromoter CDS Terminator

transcription

Genomic DNA

mRNA

protein

UTR UTR

translation


Rho-Independent Transcription Termination

(depends on DNA sequence - NOT a protein factor)

Stem-loop structure

When a nascent RNA transcript contains a series of U residues at the 3’ end preceeded by a GC rich self complementary sequences the complementary sequences base pair with one another, forming a stem loop structure.

This stem loop structures interacts with the surface of RNA polymerase causing it to pause. During this time the U-A base pairs at the 3’ end of RNA chain ( which are extremely unstable) melt releasing the RNA from the transcription complex to terminate transcription

Rho independent transcription termination


Rho independent transcription termination


The termination function of factor

The factor, a hexamer, is a ATPaseand a helicase.


Rho-Dependent Transcription Termination(depends on a protein AND a DNA sequence)

G/C -rich site

RNAP slows down

Rho helicasecatches up

Elongating complex is disrupted

1) Rho binds a stretch of GC rich sequence of nacent RNA upstream of the terminator.

2) Rho acts as hexamer, breaks ATP and with the energy moves through RNA to catch DNA-RNA hybrid and polymerase complex and terminates transcription.Copyrights@ Prof.A.K.Ghosh


Regulation of transcription in prokaryotes

•Gene regulation has been well studied in E. coli. Although there are lot of genes are present but they are not all expressed all the time. it is determined by the growth status of the cell, metabolic condition etc.

• As an example, When a bacterial cell encounters a potential food source it will manufacture the enzymes necessary to metabolize that food.

•In 1959 Jacques Monod and Fracois Jacob looked at the ability of E. coli cells to digest the sugar lactose

•In the presence of the sugar lactose, E. coli makes an enzyme called beta galactosidase to break down the sugar lactose so that E. coli can digest it for food but not in the absence of lactose

•It is the lacZ gene in E coli that codes for the enzyme β-galactosidase and this gene is present in lac operon (cluster of genes transcribed by same promoter as polycistronic mRNA)Copyrights@ Prof.A.K.Ghosh

Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements

lacI

Regulatory gene

lacZ lacY lacA DNA

m-RNA

β-GalactosidasePermease

Transacetylase

Protein

Structural GenesCis-acting elements

PlacI PlacOlac

The Lac operonCopyrights@ Prof.A.K.Ghosh

A cis-regulatory element

• cis-element is a region of DNA or RNA that regulates the expression of genes located on that same molecule of DNA (often a chromosome).

• This term is constructed from the Latin word cis, which means "on the same side as". A cis-element may be located upstream of the coding sequence of the gene it controls (in the promoter region or even further upstream), in an intron, or downstream of the gene's coding sequence, either in the untranslated or untranscribed region.


1. When lactose is absent

• A repressor protein is continuously synthesised. It sits on a sequence of DNA just in front of the lac operon, the Operator site

• The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing

Regulator gene

lac operonOperator site

z y aDNA

I O

Repressor protein

RNA polymeraseBlocked

© 2007 Paul Billiet ODWSCopyrights@ Prof.A.K.Ghosh

2. When lactose is present

• A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site)

• This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site

Promotor site

z y aDNA

I O

© 2007 Paul Billiet ODWSCopyrights@ Prof.A.K.Ghosh

Eukaryotic Transcription is Complicated

Three different polymerases:RNA polymerase I: synthesizes rRNA in the nucleolus. RNA polymerase II: synthesizes mRNA in the nucleoplasm. RNA polymerase III: synthesizes tRNA, 5S rRNA, small RNAs in the nucleoplasm

All eukaryotic RNA polymerases have 12-16 subunits (aggregates of >500 kD). Some subunits are common to all three RNA polymerases such as TBP.

Multiple promoter types :TATA Box, Initiator elements, CpG island for pol I), core elements, upstream core elements (pol I), (A box, B Box, C Box for pol III)

•Each RNA polymerase recognizes its own promoter•Many proteins (transcription factor) are involved in promoter recognition by RNA Polymerase

Eukaryotic Transcription


6

Eukaryotic Gene Structure5’ - Promoter Exon1 Intron1 Exon2 Terminator – 3’

UTR splice splice UTR

transcription

translation

Poly A

protein

Eukaryote Promoter (Pol II)


Transcription by Polymerase II

Three Steps:

Intiation:

Binding of transcription factors and Pol II to promoter,

DNA strand separation and beginning of RNA synthesis.

Elongation:

Continuous Process of RNA synthesis by RNA pol II.

Termination:

Ending of transcription after transcribing a polyA signal sequence.


TranscriptionInitiation: Assemblyof the initiationmachinery

TFIIDABFEH+RNAPII+DNACopyrights@ Prof.A.K.Ghosh


In eukaryotes, the primary transcript (pre mRNA) must be modified by:

– addition of a 5’ cap– addition of a 3’ poly-A tail– removal of non-coding sequences (introns-non

coding sequence) and joining of coding sequences (Exons) by splicing through the formation of spliceoome (with the help of snRNPs)

Post–transcriptional modification of Pre-mRNA


ppp5'NpNp

pp5'

NpNp

GTP

PPi

G5'

ppp5'

NpNp

methylating at G7

methylating at C2' of the first and second nucleotides after G

forming 5'-5' triphosphate group

removingphosphate group

m7GpppNpNp

m7Gpppm2'Npm2'Np

Pi

Capping at 5’ end of mRNA


Mature RNA

View the iActivityFor this chapter!


lariat

U pA G pU5' 3'5'exon 3'exon

intron

pG-OH

pGpA

GpU 3'U5' OH

first transesterification

Twice transesterification

second transesterification

U5' pU 3'

pGpA

GOH

5'

3'

Splicing mechanism


alternative splicing


DNA

Cytoplasm

Nucleus

Eukaryotic Transcription

ExportG AAAAAA

RNA

Transcription

Nuclear pores

G AAAAAA

RNAProcessing

mRNA


Fig. 18.17, Model of glucocorticoid steroid hormone regulation.

Eukaryotic gene regulation


Translation is the process of decoding a mRNA molecule into a polypeptide chain or protein

Translation: Protein synthesis


•Transcription and translation in eukaryotic cells are separated in space and time. Extensive processing of primary RNA transcripts in eukaryotic cells.


Translation

It is process of protein synthesis (assembly of amino acids) using mRNA as template with the help of tRNA and ribosomes (rRNA with several protein).

Therefore it requires the participation of multiple types of RNA:

• messenger RNA (mRNA) carries the information from DNA that encodes proteins

• ribosomal RNA (rRNA) is a structural component of the ribosome

• transfer RNA (tRNA) carries amino acids to the ribosome for translation


The Genetic Code

The genetic code is the way in which the nucleotide sequence in nucleic acids specifies the amino acid sequence in proteins.

A codon is a set of 3 nucleotides that specifies a particular amino acid.

Therefore, mRNA carries information from DNA in a three letter genetic code.

• A three-letter code is used because there are 20 different amino acids that are used to make proteins.

• If a two-letter code were used there would not be enough codons to select all 20 amino acids.

• That is, there are 4 bases in RNA, so 42 (4x 4)=16; where as for 3 lettered code the number is 43 (4x4x4)=64.


SU

GA

R-P

HO

SP

HA

TE

BA

CK

BO

NE

B A

S E

S

H

PO

O

HO

O

O

CH2NH2N

NH

N

N

HOH

P

O

O

HO

O

O

CH2

NH2

N

N

N

N

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

A Codon

GuanineGuanine

AdenineAdenine

AdenineAdenine

Arginine


GENETIC CODE

Therefore, there is a total of 64 codons with mRNA, 61 specify a particular amino acid.

The remaining three codons (UAA, UAG, & UGA) are stop codons, which signify the end of a polypeptide chain (protein).

This means there are more than one codon for each of the 20 amino acids. Besides selecting the amino acid methionine, the codon AUG also serves as the “initiator” codon, which starts the synthesis of a protein


mRNA contains codons which code for amino acids.


tRNA - Transfer RNA.

Each tRNA molecule has 2 important sites of attachment.

•Anticodon that binds to the codon on the mRNA molecule.

•Another site attaches to a particular amino acid.

During protein synthesis, the anticodon of a tRNA molecule base pairs with the appropriate mRNA codon.

tRNA Activation


tRNA Structure

Aminoacyl tRNA synthetase

There are 20 different aminoacyl tRNA synthetases, one for each amino acid.


Ribosome• Are made up of 2 subunits, a large one and a smaller one, each subunit contains ribosomal RNA (rRNA) & proteins.

• Protein synthesis starts when the two subunits bind to mRNA.


The ribosome has multiple tRNA binding sites:– P site – binds

the tRNA attached to the growing peptide chain

– A site – binds the tRNA carrying the next amino acid

– E site – binds the tRNA that carried the last amino acid


Translation has 3 Steps, Each Requiring Different Supporting Proteins

• Initiation– Requires Initiation Factors

• Elongation– Requires Elongation Factors

• Termination– Requires Termination Factor


Initiation:

1. Binding of initiation factors to small subunit.

2. Binding of first tRNA and mRNA to small subunit.

3. Binding of large subunit.


N-Formylmethionine (fMet)fMet is a proteinogenic amino acid.

It is a derivative of the amino acid methionine in which a formyl group has been added to the aminogroup.

It is specifically used for initiation of protein synthesis from bacterial and organellar genes, and may be removed post-

translationally.fMet is coded by the same codon as methionine, AUG. However, AUG is also

the translation initiation codon. When the codon is used for initiation, fMet is used instead of methionine, thereby

forming the first amino acid of the nascent peptide chain. When the same codon appears later in the mRNA, normal methionine is used.

Many organisms use variations of this basic mechanism.

The addition of the formyl group to methionine is catalyzed by the enzyme methionyl-tRNA formyltransferase. This modification is done after

methionine has been loaded onto tRNAfMet byaminoacyl-tRNA synthetase.


Overview of Prokaryotic Translation



Summary


Recombinant DNA

• Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other

• DNA fragments are usually derived from different biological sources

• A series of procedures used to recombine DNA segments and are called Recombinant DNA Technology


History of recombinant DNA technology

Recombinant DNA technology is one of the recent advances in

biotechnology, which was developed by two scientists named Boyer and

Cohen in 1973.


DNA recombinant technology


Basic principle of recombinant DNA technology

One DNA molecule (called insert) is isolated from one sources and then this DNA is inserted into another DNA molecule called ‘vector’

Mostly bacterial plasmid is used as vector

The recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced


Basic steps inRecobinant DNA Technology

1. Isolate the gene

2. Insert it in a host using a vector (plasmid)

3. Produce as many copies of the host as possible

4. Separate and purify the product of the gene


Step 1: Isolating the gene


Step 2: Inserting gene into vector

• Vector – molecule of DNA which is used to carry a foreign gene into a host cell.

• Bacterial plasmid is used as vector



Step 3: inserting vector into host



Applications of Recombinant DNA

Technology Large-scale production of human proteins by genetically engineered bacteria.

Such as : insulin, Growth hormone, Interferons and Blood clotting factors (VI I I & IX)


Production of Human Insulin(???)

1) Obtaining the human insulin gene Human insulin gene can be obtained by making a complementary DNA (cDNA) copy of the messenger RNA (mRNA) for human insulin.


2)J oining the human insulin gene into a plasmid(? ? ) vector

The bacterial plasmids and the cDNA are mixed together. The human insulin gene

(cDNA) is inserted into the plasmid through complementary base pairing at sticky ends.


3)Introducing the recombinant DNA plasmids into bacteria

The bacteria E.coli is used as the host cell. I f E.coli and the recombinant plasmids are mixed together in a test-tube.


4)Selecting the bacteria which have taken up the correct piece of DNA

The bacteria are spread onto nutrient agar. The agar also contains substances such as an antibiotic which allows growth of only the transformed bacteria.


Thank you


Sigma factor (): subunit of RNA polymerase that recognizes and binds to the promoter

+ polymerase= holoenzyme

1. Holoenzyme is formed and factor interacts and binds to the promoter region

2. Polymerase unwinds DNA 3. Transcription begins4. After ~10 nt are synthesized,

factor is released and polymerase undergoes conformational change.Elongation mode begins;RNA strand exits polymerase

5. Elongation6. Polymerase encounters

termination signal7. Full length RNA is released

Prokaryotic RNA polymerase and transcription


Simultaneous Transcription of a Gene by Multiple Molecules of RNA

Polymerase

Fine threads= newly synthesized transcriptsDots along DNA = RNA polymerase molecules

What is the direction of transcription?


Gene Structure:Polycistronic vs monocistronic

• Cistron: an old name for a gene• Polycistronic: 1 promoter directs

synthesis of 1 mRNA that can be translated to more than one polypeptide– Prokaryotic genes

• Monocistronic: 1 promoter directs synthesis of 1 mRNA that usually translates to only 1 protein– Eukaryotic genes


http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.html


AE

Large subunit

P

Small subunit

Translation - Initiation

fMet

UACGAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA

3’


AE

Ribosome P UCU

Arg

Aminoacyl tRNA

PheLeu

Met

SerGly

Polypeptide

CCA

Translation - Elongation

GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA

3’


AE

Ribosome P

PheLeu

Met

SerGly

Polypeptide

Arg

Aminoacyl tRNA

UCUCCA



3’


AE

Ribosome P

CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide



3’


AE

Ribosome P


Aminoacyl tRNA

CGA

Ala

CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide


3’


AE

Ribosome P


CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide

CGA

Ala


3’


Similarities between DNA Replication and DNA

Transcription

Before we begin our discussion on prokaryotic transcription, it is helpful to first point out some

similarities and differences between the process of DNA replication and DNA transcription. The processes that synthesize DNA and RNA are similar in that they use similar nucleotide building blocks. They also use the same chemical method of attack by a terminal -OH

group of the growing chain on the triphosphate group of an incoming nucleotide. Both replication and

transcription are fueled by the hydrolysis of the pyrophosphate group that is released upon attack. There

are, however, a number of important differences between these two distinct processes.


The Structure of RNA Polymerase

• There are two main segments of the RNA polymerase molecule: the core enzyme, and the sigma subunit. These two pieces are together referred to as the "holoenzyme". The core enzyme is itself composed of a beta, beta prime, and two alpha subunits; together the core is responsible for carrying out the polymerization or synthesis of RNA.

• The sigma (σ) subunit of RNA polymerase is the part of the enzyme responsible for recognizing the signal on the DNA strand that tells the polymerase to begin synthesizing RNA.

• It is through this sigma (σ) sub-unit that RNA polymerase is able to initiate transcription.


Differences between Replication and Transcription

• One major difference rests on the fact that while DNA replication copies an entire helix, DNA transcription only transcribes specific regions of one strand of the helix. During DNA transcription, only short stretches (about 60 base pairs) of the template DNA helix are unwound. As the RNA polymerase transcribes more of the DNA strand, this short stretch moves along with the transcription machinery. This process is different from that in DNA replication in which the parent helix remains separated until replication is done.

• There are slight differences in the substrates that are used in DNA replication versus transcription. Recall the structural differences between DNA and RNA. RNA's nucleotides are not deoxyribonucleotide triphosphates as in DNA. Instead, they are simply ribonucleotide triphosphates, meaning they do not lack an -OH group. Additionally, in RNA the thymine base is replaced with the base uracil. Both of these differences can be seen in DNA transcription.

• Another major difference is that DNA replication is a highly regulated process that only occurs at specific times during a cell's life. DNA transcription is also regulated, but it is triggered by different signals from those used to control DNA replication.

• One final difference lies in the capabilities of RNA polymerase versus DNA polymerase. Remember that a key problem in DNA replication lay in the initiation of the addition of nucleotides. RNA primers are needed to begin replication because DNA polymerase is unable to do it alone. DNA transcription does not have the same problem because RNA polymerase is capable of initiating RNA synthesis. The structure of the RNA polymerase is necessary for understanding all of the processes that underlie initiation, elongation, and termination and also explain some of its added capabilities.


The Start Site and the Promoter Region

• In prokaryotic cells, free RNA polymerase molecules are constantly colliding with DNA helices. The collision leads to a weak association between the DNA and RNA polymerase, which is soon broken. However, when the RNA polymerase binds to a specific sequence on the DNA, it binds tightly, forming a DNA/RNA polymerase complex. This specific site of binding is called the start site. The start site represents the location on the DNA that marks where the first nucleotide of an RNA chain should go; that spot is designated as the "plus one position". Positions that are designated as downstream in the RNA are positively numbered according to their relative position to the plus one position. All positions designated as upstream of the start site are labeled with negative numbers according to their position relative to the start site. Sequences located just upstream of the start site, called the promoter region, contain the information that signals the RNA polymerase to start transcription.


The Structure of the Promoter Region

• There are a number of key features to the promoter region that give it the ability to provide the signal initiating transcription. While nearly all promoters vary slightly, they all have general traits that can be identified. Located approximately 10 and 32 base pairs upstream of the start site are two such regions, called the -10 and -35 sequences. Each sequence consists of six base pairs. For an ideal promoter, the sequence is TTGACA for the -35 region and TATAAT for the -10 region.

• Figure %: Traditional Promoter Region• In addition to the specificity of the bases in these sequences, the spacing

between the two is also important. Ideally, this gap is 17 base pairs long. Deviations from this spacing have significant effects on the strength of the promoter region. The closer a promoter region is to matching this canonical promoter sequence, the greater its strength.

• There is a third promoter element that is sometimes seen in very strong promoters which is called the UP element. It usually is composed of alternating stretches of 5 adenine and thymine bases. It is located upstream of the -35 region.


Recognition of the Promoter Region

• RNA polymerase binds to the DNA helix at the start site. Bound to DNA, it covers a 60 base pair region within which it scans for the -35 and -10 promoters. Initially, the polymerase, and specifically the sigma subunit, binds in what is called a "closed complex" to the DNA. The RNA polymerase/promoter complex then undergoes a conformational change that breaks a number of base pairs extending from the -10 region to create a bubble in which the two DNA strands have separated. This bubble is usually approximately 17 base pairs in length. This new formation is called the "open complex". RNA synthesis is then initiated using one of the DNA strands as a template for adding complementary RNA base pairs. Transcription is usually initiated with a purine, rather than pyrimidine, base. Once initiated, the RNA polymerase moves down the DNA strand in the elongation process, which is covered in the next section.

Prokaryotic DNA Transcription Elongation and TerminationElongationThe elongation phase of transcription refers to the process through which nucleotides are added to the growing RNA chain. As the RNA polymerase moves down the DNA template strand, the open complex bubble moves also. The bubble is of a fixed number of nucleotides, meaning that at the leading end of the bubble the DNA helix is being unwound, while at its trailing end the single strands are being rejoined. Whereas separation of the DNA helix is permanent in replication, it is only temporary in transcription. depicts the beginning steps in transcription up to elongation and the relative positions of the bubble and the polymerase holoenzyme.


Prokaryotic DNA Transcription Elongation and TerminationElongation

• The elongation phase of transcription refers to the process through which nucleotides are added to the growing RNA chain. As the RNA polymerase moves down the DNA template strand, the open complex bubble moves also. The bubble is of a fixed number of nucleotides, meaning that at the leading end of the bubble the DNA helix is being unwound, while at its trailing end the single strands are being rejoined. Whereas separation of the DNA helix is permanent in replication, it is only temporary in transcription. depicts the beginning steps in transcription up to elongation and the relative positions of the bubble and the polymerase holoenzyme.


• http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html


Documents

Transcription & Translation