Overview of Transcription, Translation and Recombinant DNA Technology

BY Anindya S. 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

Organization of DNA molecule

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)

Consult: http://www.biology.arizona.edu/biochemistry/activities/DNA/04q.html

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
amino group

Formation of the peptide bond

O R1 OH NH2
O R1 OH NH2 HO R2 H2N O
R2 OH NH2 O

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

O R1 NH NH2 HO O R2 H2O

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

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

Prokaryotic promoter
5' -50 3' -40 -30 -20 -10 1 10 3' 5'

-35 region T T G A C A A A C T G T

-10 region T A T A A T A T A T T A (Pribnow box)

start

Consensus sequence

Stages of Transcription
Chain Initiation Chain Elongation Chain Termination

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

initiation

Closed complex formation

RNAP bound -40 to +20

Open complex formation

RNAP unwinds from -10 to +2

Binding of 1st NTP

Requires high purine [NTP]

Addition of next NTPs

Requires lower purine [NTPs]

Dissociation of sigma

After RNA chain is 6-10 NTPs long

RNA Synthesis is in the 5 to 3 Direction

RNA strand DNA strand

Subsequent hydrolysis of PPi drives the reaction forward

OH

OH

RNA has polarity (5 phosphate, 3 hydroxyl)

RNA polymerase
elongation

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.

12-""

Transcription Termination
Transcription ends after a terminator is transcribed

Two types of terminators in bacteria:

Rho-dependent terminators

Rho-independent terminators

Rho independent transcription termination

When a nascent RNA transcript contains a series of U residues at the 3 end proceeded 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

(depends on DNA sequence - NOT a protein factor)

Stem-loop structure

Rho independent transcription termination

Rho-Dependent Transcription Termination

(depends on a protein AND a DNA sequence)
1) Rho binds a stretch of GC rich sequence of nacent RNA upstream of the terminator.

G/C -rich site

2) Rho acts as hexamer, breaks ATP and with the energy moves through RNA to Rho helicase catch DNA-RNA catches up hybrid and polymerase complex and terminates Elongating complex is disrupted transcription.

RNAP slows down

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 -galactosidase and this gene is present in lac operon (cluster of genes transcribed by same promoter as polycistronic mRNA)

Lactose operon: a regulatory gene

Regulatory gene Cis-acting elements
lacI lacZ

and 3

stuctural genes, and 2 control elements

Structural Genes
lacY
lacA

DNA m-RNA

PlacI

Plac Olac

Protein
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 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

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.

Transcription Initiation: Assembly of the initiation machinery

TFIIDABFEH+RNAPII+DNA

Posttranscriptional 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

View the iActivity For this chapter!

Mature RNA

Splicing mechanism

lariat

alternative splicing

Eukaryotic Transcription
Cytoplasm
DNA

Transcription
RNA

RNA Processing
mRNA G
AAAAAA

AAAAAA

Nucleus

Export

Eukaryotic gene regulation

Fig. 18.17, Model of glucocorticoid steroid hormone regulation.

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

Translation: Protein synthesis

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
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.

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

Adenine

O HO P O

H O N N NH N NH2

O
CH2

Guanine Arginine

O HO P O CH2 O O

H
NH2 N N N N

Adenine

OH

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

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 expt. one amino acid is radioactively labeled and rest 19 are unlabeled and the reaction mixture are passed through filter. Since, ribosomes binds to filter if the added trinucleotide caused the labeled aminoacyl tRNA to the ribosome, then radioactivity would be detected on the filter (positive test) otherwise label will pass through-a negative test-bind filter

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.

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

A
3

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

A
3

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

A
3

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

A
3

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

A
3

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

A
3

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

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