Overview of Transcription, Translation and Recombinant DNA Technology

BY Prof. Ananta K. Ghosh

Department of Biotechnology Indian Institute of Technology Kharagpur

Eukaryotic cells with a nucleus

Prokaryotic cells without a nucleus

Nucleus Mitochondria Chloroplast Ribosomes RER SER Golgi body Cytoplasm Vacuoles

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

Macromolecules
Protein Nucleic acids olygosaccharides Lipids Complex macromolecules

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

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

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)

RNA Structure
More 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

Proteins
Proteins are made up of one or more polypeptide. Each polypeptide is a chain of co-valently bonded amino acids
The general molecular formula of an amino acid is RCH(NH2)COOH

O C

carboxylic acid group

Side chain: R characterises the amino acid

C N

H
amine group

Formation of the peptide bond

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

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

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

Y V S G A

The levels of protein structure

The Central Dogma of Molecular Biology

Cell

Transcription

DNA mRNA

Translation

Ribosome

Polypeptide (protein)

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).

TRANSCRIPTION AND TRANSLATION: Prokaryotic vs Eukaryotic

COUPLED

SEPARATE COMPARTMENTS

Gene transcription in prokaryotes

P ro k ary o tic G en e S tru ctu re
P ro m o ter UTR CDS UTR T erm in a to r

G e n o m ic D N A tran sc riptio n m RNA tran slatio n

p ro tein
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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

Promoter is a DNA sequence usually present upstream of coding regions where RNA polymerase binds to initiates transcription. UTR (Untranslated sequences): 5 UTR contains ribosome binding sites for protein synthesis; 3UTR helps in stability of RNA CDS: coding sequences for protein synthesis Terminator: Sequence for ending RNA synthesis

Requirement for transcription in prokaryotes

Gene or DNA to be transcribed, RNA polymerase, rNTPS and cellular environment

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

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 transcription

Identification of promoter by DNase 1 footprinting technique

1. A DNA fragment is labeled at one end with 32P (red dot). 2. Portions of the sample then are digested with DNase I in the presence and absence of RNA polymerase holoenzyme. 3. A low concentration of DNase I is used so that on average each DNA molecule is cleaved just once (vertical arrows). 4. The two samples of DNA then are separated from protein, denatured to separate the strands, and electrophoresed. The resulting gel is analyzed by autoradiography, 5. The set of DNA fragments left after DNase I digestion reveals the promoter

P ro m o te r
P ro m o te rs s e q u e n c e s c a n v a ry tre m e n d o u s ly . R N A p o ly m e ra s e re c o g n iz e s h u n d re d s o f d iffe re n t p ro m o te rs

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Stages of Transcription
Chain Initiation Chain Elongation Chain Termination

Bacterial Transcription Initiation

Promoter

recognition by RNA polymerase

Formation of Transcription Bubble by unwinding DNA strands Addition and Bond creation between rNTPs to start RNA synthesis Escape of transcripton apparatus from promoter (promoter clearance)

How RNA Polymerase finds promoter and Initiates Transcription

Core enzyme has the ability to synthesize RNA on a DNA template but cannot 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 -50 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

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.

Transcription Termination
Transcription ends after a terminator is transcribed
P ro k a ry o tic
P r o m o te r U T R C D S U T R

G e n e

S tru c tu re
G e n o m ic D N A

T e r m in a to r

tra n s c rip tio n m R N A

tra n s la tio n

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Two types of terminators in bacteria:

Rho-dependent terminators

Rho-independent terminators

Rho independent transcription termination

When a sequence of DNA is transcribed whose nacent RNA transcript contains a series of U residues at the 3 end proceedded by a GC rich self complementary sequences, the complementory 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 rU-dA 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

1) Rho binds a stretch of GC rich 72 nt long sequence of nacent RNA upstream of the terminator. 2) Rho acts as hexamer, after binding to RNA breaks ATP by its ATPase activity and with this energy moves through RNA to catch DNA-RNA hybrid and Unwinds by its hellicase activity and terminates transcription.

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 breaks down the sugar lactose so the E. coli can digest it for food but not in the absence of lactose It is the LAC Z gene in E coli that codes for the enzyme beta galactosidase and this gene is present in lac operon ( cluster of genes transcribed by same promoter as polycistronic mRNA)

Lactose operon: a regulatory gene and

Regulatory gene Cis-acting elements
lacI lacZ

3 stuctural genes, and 2 control elements

Structural Genes
lacY
lacA

DNA m-RNA Protein

PlacI

Plac Olac

Transacetylase -Galactosidase Permease The LAC operon

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
Repressor protein RNA polymerase

Blocked

DNA

O
Operator site

y lac operon

Regulator gene
2007 Paul Billiet ODWS

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

DNA I
2007 Paul Billiet ODWS

z y Promotor site

Eukaryotic Transcription
Eukaryotic Transcription is Horribly 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

E u k a ry o tic G e n e S tru c tu re
5 - P r o m o te r U T R E xon1 s p lic e In tr o n 1 E xon2 s p lic e T e r m in a to r 3 U T R

tra n s c rip tio n

P o ly A

tra n s la tio n

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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.

Post transcriptional modification of Pre-mRNA

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)

Capping at 5 end of mRNA

Splicing mechanism
U-rich small nuclear RNA in association of proteins called snRNP forms spliceosome and help in the splicing process.

alternative splicing

Eukaryotic Transcription
Cytoplasm
DNA

Transcription
RNA

RNA Processing
mRNA G
AAAAAA

AAAAAA

Nucleus

Export

Eukaryotic gene regulation

 http://wwwclass.unl.edu/biochem/gp2/m_biology/ani mation/gene/gene_a2.html

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

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

Translation Translation or protein synthesis 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 43 (4x4x4)=64.

A Codon
OH HO P O CH2 O O N N N NH2 N

Adenine

O HO P O CH2 O O

H O N N N NH NH2

Guanine Arginine Adenine

O HO P O CH2 O O

H NH2 N N N N

OH

GENETIC CODE 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

Deciphering the Genetic code

Marshall Nirenberg, Khorana and their collegous deciphered the genetic code by adding homopolymers such as UUU, AAA, CCC, or co-polymers such as ACA, CAA, AAC of synthetic nucleotide triplets to cell extracts containing 20 amino acyl tRNA which are capable of limited translation In each extarct one amino acid is radioactively labelled and rest 19 are unlabelled and the reaction mixture are passed through filter. Since ribosomes binds to filter, if the added trinucleotide caused the labelled aminoacyl trNA to attach to the ribosome, then radioactivity would be detected on the filter ( positive test) otherwise label will pass thrrough-a negative test.

mRNA contains codons which code for amino acids.

tRNA - Transfer RNA.

Each tRNA molecule folds as cloverleaf structure and has 2 important sites of attachment. One site, called the anticodon, binds to the codon on the mRNA molecule. The other site attaches to a particular amino acid. During protein synthesis, the anticodon of a tRNA molecule base pairs with the appropriate mRNA codon.

tRNA Structure
Aminoacyl tRNA synthetase

There are 20 different anminoacyl 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.

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.

Elongation: 1. Binding of next tRNA using EFs at A site.

EPA EPA

2. Peptide Bond formation between 2 amino acids. 3. Translocation of ribosome.

EPA

EPA EPA

Termination: 1. Binding of Release Factor to Stop Codon UGA, UAA, UAG. 2. Disassembly

Overview of Prokaryotic Translation

Translation - Initiation

fMet

Large subunit

UAC 5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

Small mRNA subunit

Translation - Elongation
Polypeptide
Met Phe Leu Ser Gly Arg

Aminoacyl tRNA

Ribosome

CCA 5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met Phe Leu Ser Gly Arg

Aminoacyl tRNA

Ribosome

CCA UCU 5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met Phe Leu Ser Gly Arg

Ribosome

CCA UCU 5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met Phe Leu Ser Gly Arg Ala

Aminoacyl tRNA

Ribosome

E
CCA

UCU 5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met Phe Leu Ser Gly Arg Ala

Ribosome

UCU CGA 5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Summary

http://wwwclass.unl.edu/biochem/gp2/m_biology/a nimation/gene/gene_a3.html

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.

Basic steps in Recobinant 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

DNA recombinant technology

Ligase

Outline of recombinant DNA Technology

Large-scale production of human proteins by genetically engineered bacteria. Such as : insulin, Growth hormone, Interferons and Blood clotting factors (VIII & IX)

Applications of Recombinant DNA Technology

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

Production of Human Insulin(???)

2)Joining 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. If 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