1) Transcription or CopyingmRNA molecules are synthesized as complementary copies of DNA template

2) TranslationConversion of genetic language in mRNA molecule (codons) into amino acid language of protein

Expression of genetic Expression of genetic informationinformation (Part II) (Part II)

Definition:Definition: a complex process in which the genetic information in DNA is a complex process in which the genetic information in DNA is decoded and used to specify the manufacture of specific protein in decoded and used to specify the manufacture of specific protein in the cell.the cell.

Includes 2 steps:Includes 2 steps:

Copy of genetic information in

mRNA (codons)

Polypeptide chain (protein)

Conversion into by

Requirements

mRNA Active tRNA Ribosomes

Translation= Protein synthesis:

tRNA (RNA polymerase III)

mRNA (RNA polymerase II)

DNA d.h.

DNA cisterone

nucleolus

Nuclear envelope

protein

Nuclear pore

tRNA

mRNA Ribosome: 2 subunits

Small subunit

large subunit

• What are the kinds of processes that take place in nucleus and cytoplasm during gene expression ?

P- site A - site

mRNAL. Subunit

S. subunit

mRNA –binding site

• RibosomeRibosome= machines of protein= machines of protein synthesis synthesis

Properties:Properties:-Large subunit:Large subunit: contains 2 binding site.contains 2 binding site. P= P= For peptidyl-tRNA (hold For peptidyl-tRNA (hold peptide chain).peptide chain). A= A= For amino-acyl-tRNA, that For amino-acyl-tRNA, that delivers the next amino acid.delivers the next amino acid.

-Small subunitSmall subunit contains mRNA binding site.contains mRNA binding site.- - Separate entities are inactiveSeparate entities are inactive..- - become active when the 2 become active when the 2 subunits are linked to mRNA subunits are linked to mRNA during translation.during translation.

-Polyribosomes Polyribosomes (polysomes) are (polysomes) are mRNA -linked clusters of mRNA -linked clusters of ribosomal molecules .ribosomal molecules .

Function: 1- translate the information in

mRNA into protein. 2- hold mRNA, amino-acyl tRNA &

polypeptide chain in a correct orientation during translation.

3- form the peptide bonds between amino acids

RibosomeRibosomestructurestructure

+

+ ATP

ADP + P

Amino-acyl – tRNA synthetase

Amino acid acceptor end

Anticodon

Amino acid

Active tRNA

5’

3’• tRNAtRNA= transfer RNA= transfer RNA

• Small molecules that are transcribed Small molecules that are transcribed from tRNA gene.from tRNA gene.

Properties:Properties:• Recognized by amino-acyl-tRNA Recognized by amino-acyl-tRNA

synthetase & ribosomes.synthetase & ribosomes.• Have amino acid acceptor 3’ end.Have amino acid acceptor 3’ end.• Have anticodon that recognizes and links Have anticodon that recognizes and links

the complementary mRNA –codon. the complementary mRNA –codon.

Types:Types: 1)1) initiator tRNAinitiator tRNA (carries methionine (carries methionine

recognizes & links the start codon; AUG). recognizes & links the start codon; AUG). 2) regular tRNA2) regular tRNA for other amino acids. for other amino acids.

• -there are -there are ((40 tRNA, 20 A.A., 61 40 tRNA, 20 A.A., 61 codonscodons).).

Structure of tRNAStructure of tRNA

1) Initiation5’ 3’

Small subunit

mRNA

5’

Small subunit

mRNA

P -site A -site

Methionine

Initiator-tRNA Start codon

Anticodon

5’

5’ 3’3’

• Steps ofSteps ofProtein SynthesisProtein Synthesis

- Begins when initiator complex (initiator tRNA & small ribosomal subunit) binds the start codon of mRNA.

- That followed by formation of functioning ribosome by binding of large ribosomal subunit to initiator complex

2) Elongation

5’

Small subunit

5’

Small subunit

5’mRNA

GTP GDP+P+E

1st step

2nd step

3rd step

1) Begins by occupying A- site by 2nd tRNA with its amino acid, that carries anticodon complementary to the next mRNA –codon.

2) Peptide bond formation; Amino acid is detached from tRNA in P-site & joined by peptide bond to amino acid linked to tRNA in A-site.

3) Translocation, Ribosome moves one codon in

direction 5’ – 3’, that leads to: - release tRNA from P-site to the cytoplasm. - transfer tRNA & the peptide chain transfer from A- site to P-site. - A- site become open to receive another suitable tRNA.

by repeating steps 1, 2, 3 (elongation cycle), the peptide chain elongates till the ribosome reaches the stop codon.

3) Termination

5’

Releasing factor

dissociation

Polypeptide chain

At stop codon:Release factor recognizes and binds the mRNA - stop codon. It terminates the protein synthesis by releasing:- Large & small ribosomal subunits- Polypeptide chain- tRNA molecule- Releasing factor- mRNA

In Eukaryotic cells:

Transcript mRNA is Transcript mRNA is immature (called pre-immature (called pre-mRNA), why?mRNA), why? a) - contains non-coding a) - contains non-coding regions (regions (intronsintrons) between ) between protein coding regions protein coding regions ((exonsexons).).

b) - needs modifications b) - needs modifications before it becomes before it becomes competent competent for transport & translation.for transport & translation.

• In prokaryotic cell (bacteria)

- translation is coupled with

transcription, Protein synthesis begins while the

mRNA is being completed, as multiple ribosomes attach to the mRNA to form a polyribosomes

- No modification or processing, why ?

• Posttranscriptional Posttranscriptional modification & processing of modification & processing of mRNA in Eukaryotic CellmRNA in Eukaryotic Cell

1) Capping:1) Capping:

- - Begins when mRNA is about 20-Begins when mRNA is about 20-30 nucleotides long.-30 nucleotides long.-

((7 –methyl guanosine cap7 –methyl guanosine cap is is added to 5’ end of mRNAadded to 5’ end of mRNA).).

Importance:Importance: 1) Non-capped mRNA can not be 1) Non-capped mRNA can not be

recognized by ribosomes.recognized by ribosomes.2) Protection against degradation.2) Protection against degradation.3) Increase the stability of mRNA3) Increase the stability of mRNA..

Half life:Half life: - about - about 10 hs10 hs. In Eukaryotic cell.. In Eukaryotic cell. - about - about 2 mins2 mins. In prokaryotic cell. In prokaryotic cell..

2) Polyadenylated tail2) Polyadenylated tail ( Poly-A tail)( Poly-A tail)

addition of addition of 100 – 250 adenine 100 – 250 adenine nucleotidesnucleotides to the 3’ end of to the 3’ end of completed mRNA.completed mRNA.

Importance:Importance:1) 1) Help in passage of mRNA from Help in passage of mRNA from

nucleus into the cytoplasm.nucleus into the cytoplasm.

2) 2) Stabilizes the mRNA against Stabilizes the mRNA against degradation (increase life span degradation (increase life span of mRNA in cytoplasm)of mRNA in cytoplasm)

• Types of modificationsTypes of modifications

3) Splicing:

= Means removing the introns (non-coding regions) and splicing the exons (coding regions) together.

• Removal of introns is performed by small nuclear ribonucleoprotein complex enzyme (snRNPC).

• Function:formation a continuous protein coding message.

-OHP-P-P-

G-P-P-P- -OH

G-P-P-P- -AAAAAA…

G-P-P-P----AAAAAA…

Intron I Intron IIImmature mRNA

1) capping

2) Addition polyadenylate tail to 3’end.

by addition 7- methyl Guanosine to 5” end.

3) Splicing the exons to form continuous coding message

Remove introns I & II

G-P-P-P----AAAAAA…

5’ 3’

Mature mRNA

Nuclear envelope

Mature mRNA becomes competent for transport & translation in cytoplasm

Gene expression inGene expression in

Eukaryotic cell prokaryotic cellEukaryotic cell prokaryotic cell

1) Transcription & 1) Transcription & translation are not translation are not coupled.coupled.

2) Average half life 10 2) Average half life 10 hourshours

(translation (translation continuous about 10 continuous about 10 hours).hours).

3) After transcription, 3) After transcription, mRNA is modified & mRNA is modified & processed.processed.

1) Transcription & 1) Transcription & translation are coupled.translation are coupled.

2) Average half life 2 2) Average half life 2 minutesminutes

3) mRNA is ready for 3) mRNA is ready for translation as soon as it translation as soon as it transcribed (no transcribed (no modification).modification).

Half life = the time that required for deterioration half of the molecule forming mRNA.

1) 1) Triplet codeTriplet code (three bases) (three bases)2) 2) UniversalUniversal for all organisms for all organisms ex. : UUU = phenylalanine in all ex. : UUU = phenylalanine in all

organisms.organisms.

3) 3) RedundantRedundant, , redundancy of redundancy of genetic codegenetic code: more than one : more than one code specify one amino acid. (61 code specify one amino acid. (61 codon, 40 tRNA, 20 amino acid).codon, 40 tRNA, 20 amino acid).

4) 4) Reads as a seriesReads as a series of nucleotide. of nucleotide. - No comma between the codon- No comma between the codon - The start codon determines the - The start codon determines the

reading frame.reading frame.

5) The codon could undergo 5) The codon could undergo mutationmutation

• Characters of genetic codeCharacters of genetic code

The Genetic CodeThe Genetic Code

Definition:Definition: Any change in the nucleotide sequence of DNA. Any change in the nucleotide sequence of DNA.

Two types:Two types: 1) 1) Unstable mutation:Unstable mutation: Revert back to original sequence.Revert back to original sequence.

2) 2) Stable mutation:Stable mutation: Change the characteristics (phenotype) ofChange the characteristics (phenotype) of organism. organism.

• MutationsMutations

Definition Definition :: An organism showing deviation in some An organism showing deviation in some characters (phenotype), whose progeny characters (phenotype), whose progeny maintain these deviances. maintain these deviances.

Cause:Cause: molecular change in hereditary material.molecular change in hereditary material.

Types:Types:1- Base substitution (Missense & nonsense mutants).1- Base substitution (Missense & nonsense mutants).2- Frame –shift mutants.2- Frame –shift mutants.

• MutantsMutants

a) Missense mutationa) Missense mutation Cause:Cause: Base substitution mutation in DNA. Base substitution mutation in DNA. Results:Results: one amino acid is replaced with another in a protein. one amino acid is replaced with another in a protein. The effect:The effect: depends upon the position of the replaced amino acid. depends upon the position of the replaced amino acid.

Missense mutation(Base substitution

mutation)

Reduce or absence the enzyme activity

( in Sickle cell disease; glutamic acid is replaced with valine in

Hemoglobin)

No change in enzyme activity

The amino acid is located at or near the active site

The amino acid is not a part of the active site

2) Missense & 2) Missense & Nonsense mutants:Nonsense mutants:

b) Non-sense mutation:b) Non-sense mutation:

Cause:Cause: mutation that creates an internal mutation that creates an internal stop codon in a genestop codon in a gene, ,

will prematurely terminates the encoded protein.will prematurely terminates the encoded protein.5’____ ATG ……………..T A C……………TAG____3’3’____ TAC …………….. AT G……………ATC____5’

5’____ ATG ……………..T A G……………TAG____3’3’____ TAC …………….. AT C……………ATC____5’

mutation

DNA d.h.

5’____ AUG …………….. UAG……………UAG____3’

transcription

Start codon internal stop normal stop codon codon

mRNA

Meth. ________________Protein terminates prematurelyshorter or nonfunctional protein

translation

Base –pair substitution leads to formation internal stop codon (UAG, UGA, UAA), that replaces the amino acid codon.

Cause:Cause: - Insertion or deletion base – pair.- Insertion or deletion base – pair. Results:Results: - Misreading of all codons from the point of deletion- Misreading of all codons from the point of deletion or insertion to the end of the message. or insertion to the end of the message. - Change the amino acid sequence of encoded polypeptide- Change the amino acid sequence of encoded polypeptide chain.chain.

AUG ACA CAA UGG ACU GAC ………… mRNA

Meth. ………………………………………… protein (normal)

AUG ACA CAU GGA CUG AC ………… mRNAMeth. ………………………………………… protein (unuseful)

A Deletion of a single base

• Results: - formation of unuseful new polypeptide chain.

3) Frame –shift mutants3) Frame –shift mutants