Lf101 lecture 7 8 - tuesday 1st april 2014 -flow of genetic information & genetic code-1

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

Lecture 7-81st April Tuesday 2014

Flow of genetic information: Three aspects -

Transcription, Translation&

Genetic code


Let’s first appreciate the problem of transfer of information from DNA

(genes) to protein


Consider the case of Sickle Cell Anemia? A serious condition in which red blood

cells can become sickle-shaped

Normal red blood cells are smooth and round. They move easily through blood vessels to carry oxygen to all parts of the body.

Sickle-shaped cells don’t move easily through blood. They’re stiff and sticky and tend to form clumps and get stuck in blood vessels.

The clumps of sickle cell block blood flow in the blood vessels that lead to the limbs and organs. Blocked blood vessel can cause pain, serious infection, and organ damage.

While the defect is seen in the protein, sickle cell anemia is produced by changes in gene sequence


Normal and Sickled Red Blood Cells in Blood Vessels

Figure A shows normal red blood cells flowing freely in a blood vessel. The inset image shows a cross-section of a normal red blood cell with normal hemoglobin.

Figure B shows abnormal, sickled red blood cells clumping and blocking the blood flow in a blood

vessel. The inset image shows a cross-section of a sickled red blood cell with abnormal strands of

hemoglobin.

Source from http://www.nhlbi.nih.gov/health/dci/Diseases/Sca/SCA_WhatIs.html


• What could be the reason(s) for the wrong message from the DNA that produced a wrong protein (hemoglobin)?

• How ‘information’ encoded in the DNA of the nucleus is transferred to the cytoplasm

• How does this information ‘translate’ into protein??

Questions?


DNA RNA protein Transcription Translation

Replication

Reverse transcription

Central dogma of Molecular biology सि�द्धां��त

We will now explore this flow of information: that is, process

of decoding the genetic message

Flow of information Nucleic acid to

protein

Flow of information: nucleic acid to nucleic

acid

Nature of information flow

Why there is no loss of

information during

replication? How much of the information in DNA goes to

RNA??

RECAP


Flow of genetic information

A gene is transcribed into RNA

which is then translated into the polypeptide

DNA

Transcription

RNA

Protein

Translation


FLOW OF GENETIC INFORMATION FROM DNA TO RNA TO PROTEIN

Gene (DNA) is a linear sequence of many nucleotides

DNA is transcribed into a linear sequence of RNA

RNA is translated into a linear sequence of amino acids – polypeptides (protein)

Entire business of flow of genetic information thus depends on constructing

(transferring information) one kind of polymer from another

DNA RNA Protein(polypeptide chain


Transcription – Step I

Template DNA Strands unzip locally

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


Transcription – Step II

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

U G C A U A G C G C A U

One of the DNA strands serve as a template and copied as mRNA

(messenger RNA)

mRNA

DNA



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

U G C A U A G C G C A U

DNA

RNA

mRNA is then releases


Chemical structure of RNA

Notice the difference between ribose and deoxyribose in the figure above. In ribose, carbon atom #2 carries one hydroxyl group (colored red). In deoxyribose, carbon atom #2 carries a hyrogen atom instead of a hydroxyl group.


Cytosine

Thymine

Uracil

Pyrimidine bases

DNA/RNA DNA RNA

In place of thymine in DNA


Transcription

RNA polymerase RNA nucleotides

Direction of transcription

Template Strand of DNA

Newly made RNA

TC

AT C C A A T

T

GG

CC

AATTGGAT

G

U

C A U C C AA

U


In the nucleus, the DNA helix unzips

And RNA nucleotides line up along one strand of the DNA, following the base pairing rules

As the single-stranded messenger RNA (mRNA) peels away from the gene

The DNA strands rejoin


Steps in transcription of a geneRNA polymerase

DNA of gene

PromoterDNA Terminator

DNA

Area shownIn Figure 10.9A

GrowingRNA

Completed RNARNApolymerase

1 Initiation

2 Elongation

3 Termination


T A C T T C A A A A T C

A T G A A G T T T T A G

A U G A A G U U U U A G

Transcription

Translation

RNA

DNA

Met Lys PhePolypeptide

Startcondon

Stopcondon

Strand to be transcribed

How does DNA/RNA code for protein?

Codon - a sequence of nucleotides that

codes for an amino acid

STEP III TRANSLATION


How can four nucleotides code for 20 amino acids?

If one nucleotide codes for one amino acid then a maximum of four amino acid could be coded

If two nucleotide code for one amino acid then a maximum of 16 amino acid could be coded

If three nucleotides code for one amino acid then a maximum of 64 codons can be generated, while the number of amino acids are only 20


The dictionary of genetic code (64 potential codons)

आनु�वा�न्शि�की की�ड की शब्दकी�श 5’-3’

A dictionary offers

translation from one

language to the other.

Which two languages are being

translated in a genetic

dictionary?

Why do we need this

translation?

Where does it take place?

What is the name of this

process?


Hallmarks of genetic code

Triplet : three consecutive nucleotides code of one amino acid

Redundant : meaning not unique (there are more than one codon for most amino acids)

Universal: All organisms bacteria to human use the same genetic codes/dictionary

Comma-less: meaning consecutive codons are readout into an amino acid sequence without gaps


How does an universal genetic code impact Biotechnology??

What would have been the case if bacterial genetic code were to be different from human?

What do we understand when we mean when we say that genetic code is universal?


DNA

Messenger-RNA

Transfer RNA

Amino acid

Key players in decoding the genetic message


Transfer RNA

Leucine

Codon

Anti-codon


How genetic code is readout in the cell


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

3’ U G C A U A G C G C A U 5’ mRNA

5’

3’

5’G U C

3’

C A G

3’5’

tRNA

Amino acid

Anti-Codon

Codon


Many amino acids are specified by more than one codon-degeneracy

Codons specifying the same amino acid are called synonyms

Connect the three points about genetic codes: redundancy, degeneracy and synonymous


CUGCUG CUCCUC

Codon-anticodon pairing of two tRNA Leu molecules

5’- 3’ 5’- 3’

Codon

GACGAC3’- 5’

Anti- codon GAGGAG3’- 5’


Three codons direct chain termination

Three codons, UAA, UAG, and UGA signify chain termination.

They are not read by tRNAs but by proteins called release factors (RF1 and RF2 in bacteria and eRF1 in eukaryotes).


Three Rules

Codons are read in a 5’ to 3’ direction.

Codons are non-overlapping and the message contains no gaps.

The message is translated in a fixed reading frame which is set by the initiation codon.


FATCATATETHERAT

Meaning and consequences of non-overlapping & comma-less reading of the triplet codons

FATATATETHERAT

Let’s consider a metaphor of a normal DNA sequence

What is the

problem here?

What is the consequenc

e?


Mutation: a change in genetic code

1. Missense mutation: An alternation that changes a codon specific for one amino acid to a codon specific for another amino acid.

2. Nonsense or stop mutation: An alternation causing a change to a chain-termination codon.

3. Frame-shift mutation: Insertions or deletions of one or a small number of base pairs that alter the reading frame.


How the genetic code was cracked?

The use of artificial mRNAs and the availability of cell-free systems for carrying out protein

synthesis began to make it possible to crack the code


Codon Assignments from Repeating Copolymers

Organic chemical and enzymatic techniques were

used to prepare synthetic poly-ribonucleotides with known

repeating sequences.


Experimental Results:

UUU codes for phenylalanine.

CCC codes for proline.

AAA codes for lysine.

The guanine residues in poly-G firmly hydrogen bond to each other and form multistranded triple helices that do not bind to ribosomes.


copolymercopolymer Codons Recognized

Codons Recognized

Amino Acids Incorporated Amino Acids Incorporated

Codon Assignment

Codon Assignment

(CU)” CUC|UCU|CUC… Leucine 5’-CUC-3’

Serine UCU

(UG)” UGU|GUG|UGU… Cystine UGU

Valine GUG

(AC)” ACA|CAC|ACA… Threonine ACA

Histidine CAC

(AG)” AGA|GAG|AGA… Arginine AGA

Glutamine GAG

(AUC)” AUC|AUC|AUC… Polyisoleucine 5’-AUC-3’

(CU)” CUC|UCU|CUC… Leucine 5’-CUC-3’

Serine UCU

(UG)” UGU|GUG|UGU… Cystine UGU

Valine GUG

(AC)” ACA|CAC|ACA… Threonine ACA

Histidine CAC

(AG)” AGA|GAG|AGA… Arginine AGA

Glutamine GAG

(AUC)” AUC|AUC|AUC… Polyisoleucine 5’-AUC-3’


What Are Mutations?What Are Mutations?

• Changes in the nucleotide sequence of DNA

• May occur in somatic cells (aren’t passed to offspring), but could cause diseases, such as cancer

• May occur in gametes (eggs & sperm) and be passed to offspring


Nature of point mutations

1. Missense mutation: An alternation that changes a codon specific for one amino acid to a codon specific for another amino acid.

2. Nonsense or stop mutation: An alternation causing a change to a chain-termination codon.

3. Frameshift mutation: Insertions or deletions of one or a small number of base pairs that alter the reading frame.


Point Mutation

Change of a single nucleotide

Includes the deletion, insertion, or substitution of ONE nucleotide in a gene


Types of Gene/Point Mutations

Substitutions

Insertions

Deletions

Frameshift


Frameshift Mutation

Inserting or deleting one or more nucleotides

Changes the “reading frame” like changing a sentence

Proteins built incorrectly


Gene Mutation Animation


Problems

THE FAT CAT ATE THE BIG RAT

THE FAT ATA TET HEB IGR AT Delete C

THE FAT ATA ATE THE BIG RAT Insert A

Frame shift mutation


RBC Structure

of Hb

Primary sequence of Hb –first 26 amino acid

RECALL


Example Point Mutation

• Sickle Cell disease is the result of one nucleotide substitution

• Occurs in the hemoglobin gene


Problem

Before the true nature of the genetic coding process was fully understood, it was proposed that the message might be read in overlapping triplets. For example, the sequence of GCAUC might be read as GCA CAU AUC

G C A U C

Think of test that would negate this possibility


Consider a gene that specified the structure of hemoglobin. Arrange the following events in the most likely sequence in which they would take place

a. Anemia is observed

b. The shape of the oxygen binding site is altered

c. An incorrect codon is transcribed into hemoglobin mRNA

d. The ovum (female gamete) receive a high radiation dose

e. An incorrect codon is generated in the DNA of the hemoglobin gene

f. A mother (an X-ray technician) accidentally steps in front of an operating X-ray generator

g. A child dies

h. The oxygen-transport capacity of the body is severely impaired

i. A wrong tRNA is attached j. Nucleotide pair substitution occurs in the DNA of the gene for hemoglobin


If an mRNA of human beta-globin gene is added to bacterial cell free extract then human beta globin polypeptide is formed (translated)What does this observation signify

with respect to genetic code?

Question


Home assignment

The amino acid sequence shown in the following table was obtained from the central region of a particular polypeptide chain in the wild

type and several mutant bacterial strains

a. Wild type phe leu pro thr val thr thr arg trp

b. Mutant 1 phe leu his his gly asp asp thr val

c. Mutant 2 phe leu pro thr met thr thr arg trp

d. Mutant 3 phe leu pro thr val thr thr arg

e. Mutant 4 phe pro pro arg

f. Wild type phe leu pro ser val thr thr arg trp

1 2 3 4 5 6 7 8 9

Codon

For each mutant, say what change has occurred at the DNA level, whether the change is a base pair substitution or frame shift mutation, and in which codon the mutation occurred


Home assignment In mutant strain X of E. coli, a leucine tRNA that recognizes the codon 5’-CUG-3’ in normal cell has been so altered that is now recognizes the codon 5’-GUG-3’.

A missense mutation (lets call this mutation Y) that affects amino acid 10 of a particular protein is suppressed in mutant X cells (that is when the cells are mutant for both X and Y, which now appears wild type)

a. What mutational even has occurred in mutant X cells?

b. What amino acid would normally be present at position 10 of the protein (without the missense mutation)?

c. What amino acid is put in at position 10 if the missense mutations is not suppressed ?

d. What amino acid is put in at position 10 if the missense mutations is suppressed ?

Lf101 lecture 7 8 - tuesday 1st april 2014 -flow of genetic information & genetic code-1

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