free molecular biology lecture notes pdf biochemistry

LECTURE NOTES ON MOLECULAR BIOLOGY

Lecture notes on DNA replication, Transcription and Translation

Vijay Marakala

1

Definition

Prokaryotic replication

Semiconservative mechanism

Basic requirements

- Substrates

- Template

- Enzymes and proteins

- Primers

Stages of replication

- Initiation

- Elongation

- Termination

Differences between prokaryotic

and eukaryotic replication

Inhibitors of DNA replication

The duplication or synthesis of

DNA is called replication

Definition: DNA REPLICATION

PROKARYOTIC REPLICATION

Basic mechanism of replication is

same both in prokaryotic and

eukaryotic DNA but replication in

prokaryotes is simpler and well

understood.

SEMICONCERVATIVE MECHANISM

Each replicated duplex daughter

DNA molecule contains one

parent strand and one newly

sythesised strand.

Dr Vijay Marakala, MBBS. M.D. Assistant professor,

Department of Biochemistry, SIMS & RC , MUKKA - SURATHKAL, MANGALORE.

[email protected]

LECTURE NOTES ON DNA REPLICATION

Please refer following

textbooks

Biochemistry by Pankaja

Naik

Textbook of Biochemistry

by DM Vasudevan

Harper’s Illustrated

Biochemistry

2

Protein or Enzyme Function

DNA A protein Opens duplex at origin of replication

DNA B protein (Helicase) Unwinds DNA Primase Synthesises RNA primer SSB(Single strand binding protein)

Binds separated single stranded DNA and stabilizes it

DNA topoisomerase I Relieves torsional strain by cutting and joining single strand

DNA topoisomerase II Relieves torsional strain by cutting and joining both strands

DNA polymerase DNA chain elongation DNA ligase Joins Okazaki fragments Ter binding protein Prevents the helicase from

further unwinding and facilitates termination

BASIC REQUIREMENTS

Substrates: dATP, dGTP, dCTP, dTTP

Template: Separated DNA strands are the template for synthesis of new

daughter strands.

Enzymes and proteins involved in replication

DNA polymerase I – Removal of RNA primer and replacing with deoxyribonucleotides

DNA polymerase II – Proofreading and DNA repair

DNA polymerase III - DNA chain elongation

There are 3 types of DNA polymerases in prokaryotes

3

Enzymes and proteins involved in replication

Primer

Primase synthesises RNA primer (in a 5’ to 3’ direction) using DNA as a

template. DNA polymerase initially adds a deoxyribonucleotide to the 3’-OH

group of the primer and then continues to add deoxyribonucleotides to the 3’-

end of the growing strand

STAGES OF REPLICATION

1. INITIATION

2. ELONGATION

3. TERMINATION

Initiation

DNA A protein recognizes and binds to

“ORI” and unwinds or separates the

DNA

DNA B protein (helicase) binds to this

region and further unwinds the DNA

and formation of replication bubble

Stress produced by unwinding is

released by topoisomerases

SSB proteins stabilizes the separated

strands and prevents reassociation.

Binding of primase results in synthesis

of RNA primer(5’ to 3’ direction) which

are complememtary to bases of DNA

“ORI” means origin of replication i.e specific sequence of DNA

DNA replication is bidirectional

DNA replication always starts from 5’ to 3’ direction

Replication bubble

4

Elongation

For animation refer DNA replication ppt

Because of antiparallel nature of two strands, the synthesis of DNA along

the two strands is different.

One strand of DNA is synthesized continuously and it is known as

leading strand.

Another strand is synthesized discontinuously in the form of okazaki

fragments. It is known as lagging strand. The length of these

segments ranges from 1000 to 2000 bases. They are also synthesized

in 5' to 3' direction only.

The gaps between okazaki fragments are filled by DNA polymerase I.

RNA primers are removed by 5' → 3' exonuclease activity of DNA

polymerase I.

Finally DNA ligase joins the ends of okazaki fragments.

5

Features Prokaryotes Eukaryotes

RNA primer length 50 nucleotides 9 nucleotides

DNA polymerase I,II,III α,β,γ,δ,ε

Number of origins Single Multiple

Nucleotide length of Okazaki 1000-2000 nucleotides 200 nucleotides

Rate of replication 500 nucleotides/sec 50 nucleotides/sec

Termination

A specific protein “ter binding protein” binds a specific sequences “ter”

sequences and prevents the helicase from further unwinding of DNA

and facilitates the termination of replication.

Differences between prokaryotic and eukaryotic replication

Inhibitors of DNA replication

Nalidixic acid

Novobiocin

Ciproploxacillin

Inhibit prokaryotic topoisomerase II.

Widely used as antibiotics for treating

urinary tract infections and other

infections.

Adriamycin

Etoposide

Doxorubicin

Inhibit eukaryotic topoisomerase

II. Widely used as anticancer drugs

6

LECTURE NOTES ON TRANSCRIPTION


Department of Biochemistry, SIMS & RC , MUKKA - SURATHKAL, MANGALORE.

[email protected]

Definition

Similarities and differences between

replication and transcription

Basic requirements for transcription

Template

Substrate

Enzyme

Stages of transcription

Initiation

Elongation

Termination

Differences between prokaryotic and

eukaryotic transcription

Post-transcriptional processing

Cleavage of precursor of RNA

Terminal addition of nucleotides

Base modification

Splicing

Inhibitors of transcription

Please refer following textbooks

Biochemistry by Pankaja Naik

Textbook of Biochemistry by DM

Vasudevan


Biochemistry

Definition

Synthesis of RNA using DNA as a template

Similarities and differences between

replication and transcription

Similarities

3 stages

Synthesis occurs in the 5’→3’

direction

Follows Watson-Crick base pairing

Differences

Ribonucleotides are used in RNA

synthesis

Uracil replaces Thymine

Primer is not required

Only a very small portion of genome is

transcribed

Basic requirements for transcription

Template

The strand of DNA that is transcribed into RNA is called as template or sense strand whereas other strand is called as coding or anti-sense strand.

For more diagram please go

through following ppt slides

Transcription


7

Substrates: ATP, GTP, CTP and UTP

Enzyme:

In prokaryotes, DNA directed RNA polymerase is the major enzyme of transcription.

It catalyzes the synthesis of all three types of RNAs like mRNA, tRNA and rRNA.

Holoenzyme consists of five subunits. They are ∝ ∝ ββ′ and σ.

The coenzyme consists of only four subunits αα ββ'

Stages of transcription Initiation

Elongation

Termination

Initiation

1. Initiation of RNA synthesis involves binding of RNA polymerase to the template strand. Certain regions of DNA serves as initiation signals. They are known as promoter sites.

2. RNA polymerase identifies promoter by virtue of σ factor. 3. Unwinding of DNA occurs and sets the stage for first phosphodiester

linkage formation.

Promoters Specific sequence of DNA functions as transcription signals. They are referred as Promoters. Usually they are located away (upstream) from start point (+1) of transcription. Two such promoters are known in prokaryotes. One promoter is located 10 nucleotides away from start site.It is known as –10 region or Pribnow or TATA box. Another promoter is located 35 nucleotides away from start site. It is known as –35 region. Usually the promoters facilitate dissociation of DNA strands so that DNA unwinds to favors transcription by RNA polymerase.

8

Elongation

Elongation of RNA molecule occurs in 5' → 3' direction as the RNA polymerase polymerizes rNTPs anti-parallel to template strand.

As RNA polymerase progress along the DNA molecule unwinding of DNA

takes place ahead of 3' end of nascent RNA. The growing RNA or nascent

RNA is base paired to template strand.

Termination

Prokaryotic termination of transcription occurs by one of the two well

characterized mechanisms.

Rho-dependent

Rho-independent

Rho-dependent Termination

Rho-dependent termination requires

a protein factor called rho whichever

recognizes then termination signal

that displaces the RNA polymerase

from template resulting in

termination of RNA synthesis.

Rho-independent Termination

Rho-independent termination

involves a secondary structure (hair-

pin loop) formed in the newly

synthesized RNA, which dislodges the

RNA polymerase from DNA template

resulting in the release of transcript.

9

Binds to tightly to DNA and prevent unwinding. Actinomycin D

Binds to β subunit of prokaryotic RNA polymerase Rifampicin

Inhibits eukaryotic RNA polymerase α-Amanitin

Differences between prokaryotic and eukaryotic transcription

The basic mechanism of transcription is same in eukaryotes as that of prokaryotes;

however eukaryotic transcription differs mainly with respect to

RNA polymerase: type I, II and III

Promoter site: -25 TATA box(Hogness box) and -75 CAAT box


All three types of RNAs are synthesized in precursor forms in eukaryotes. These precursors are converted to functional RNA molecules by post-trasncriptional modifications. Usually, these modifications takes place in nucleus. Some prokaryotic RNAs also undergo these modifications.(prokaryotic mRNA is not processed post-transcriptionally)

Cleavage of precursor of RNA

Terminal addition of nucleotides

Base modification

Splicing

mRNA Processing

1. Capping at the 5’ end

2. Addition of poly-A tail at the 3’ end

3. Splicing to remove intron

tRNA Processing

1. Cleavage of 5’ leader sequence

2. Splicing

3. Replacement of 3’ UU by CCA

4. Modifications of several bases

rRNA Processing

Ribosomal RNAs are synthesized in large precursor form known as pre rRNA

Inhibitors of Transcription

10

LECTURE NOTES ON TRANSLATION [protein biosynthesis]


Department of Biochemistry, SIMS & RC, MUKKA - SURATHKAL, MANGALORE.

[email protected]

Definition

Basic requirements

mRNA to be translated

tRNAs

Ribosomes

Energy in the form of ATP and

GTP

Enzymes and specific factors

Stages of translation

Activation of amino acids

Initiation

Elongation

Termination

Post-translational modification

Proteolytic cleavage

Modification of amino acids

Subunit aggregation

Protein folding and chaperones

Inhibitors of translation

Please refer following

textbooks

Biochemistry by Pankaja Naik

Textbook of Biochemistry by

DM Vasudevan


Biochemistry

Definition

Process by which ribosomes convert the

information carried by mRNA in the form of

genetic code to the synthesis of new

protein.

Basic requirements


tRNAs

Ribosomes

Energy in the form of ATP and GTP

Enzymes and specific factors

Stages of Translation


Initiation

Elongation

Termination


For protein synthesis, initial activation of amino acid is required. This activation is essential because energy is required for peptide bond formation. Activation involves esterification of amino acid with tRNA.

11

Initiation

Basic requirements for Initiation

Ribosome


The initiating Met-tRNAi met

Initiation factors – Prokaryotes-IF1, IF2 &IF3. Eukaryotes-EIF1 to EIF9

Steps involved in Initiation

Ribosomal dissociation

Formation of 43S pre-initiation complex

Formation of 48S initiation complex

Formation of 80S initiation complex

12

Elongation

Basic requirements for Elongation

Various elongation factors –for Prokaryotes(EF), for Eukaryotes- eEF

80S initiation complex

The next aminoacyl tRNA specified by the next coding triplet in mRNA

Steps involved in Elongation

Binding of next aminoacyl tRNA specified by the next coding triplet

in mRNA to ‘A’ site

Formation of peptide bond

Translocation

Formation of peptide bond Two amino acyl-tRNAs on the two sites of ribosome sets the stage for first peptide bond formation. The peptidyl transferase activity of 60S ribosomal subunit catalyzes the peptide bond formation between two amino acids. This process is also known as trans-peptidation, because the peptide bond formation involves transfer of met from tRNA located in the P site to α-amino group of amino acyl-tRNA in the A site. Nucleophilic attack of α-amino group of incoming amino acyl-tRNA on carboxyl group of met of met tRNA generates peptide bond. As a result, a dipeptide is generated on tRNA of A site leaving empty tRNA f on P site

13

Termination

The elongation steps are

repeated until one of the three

(UAA, UAG, UGA) termination

or nonsense codons of mRNA

appear in the ‘A’ site.

Once the ribosome reaches a

termination codon, releasing

factors are capable of

recognizing the termination

signal present in the ‘A’ site.

Prokaryotes have 3 release

factors-RF1, RF2 &RF3

Eukaryotes have only one

release factor eRF.

Post-translational modification

In order to achieve native biologically active form of polypeptide, it must

undergo processing and folding in proper three dimensional conformation.

These alterations are known as post-translational modifications.

Proteolytic cleavage


Subunit aggregation


14


SUBUNIT AGGREGATION


Proper folding of protein into its

three dimensional confirmation is

assisted by chaperones. Improper

folding leads to inactive protein or

sometimes may cause diseases.

Inhibitors of Translation

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