Management of Genetic

Information

Learning objectives

Understand the mechanism of DNA

replication, RNA synthesis and protein

synthesis

Flow of genetic information

Two possible models of the DNA

replication

Expt by Meselson-Stahl proved the

semiconservative model of replication

Which direction does replication go?

Major enzyme: DNA polymerase III DNA double helix unwinds at a specific point called an

origin of replication

Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is bidirectional in most organisms

At each origin of replication, there are two replication forks, points at which new polynucleotide chains are formed

There is one origin of replication and two replication forks in the circular DNA of prokaryotes

In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks at each origin

Replication in

prokaryotes

Replication in

eukaryotes

DNA synthesis based on two template strands: leading strand and

lagging strand templates; mechanism in prokaryotes is presented

DNA is synthesized from its 5’ -> 3’ end (from

the 3’ -> 5’ direction of the template)

the leading strand is synthesized continuously in

the 5’ -> 3’ direction toward the replication fork

the lagging strand is synthesized

semidiscontinuously (Okazaki fragments) also in

the 5’ -> 3’ direction, but away from the replication

fork

lagging strand fragments are joined by the

enzyme DNA ligase

Replication fork

Enzymes and proteins in DNA replication

The action of DNA polymerase

Why 53’ direction?

Start of DNA replication

Unwinding DNA gyrase introduces a swivel point in

advance of the replication fork

a helicase binds at the replication fork and

promotes unwinding

single-stranded binding (SSB) protein protects

exposed regions of single-stranded DNA

Primase catalyzes the synthesis of RNA primer

Synthesis

catalyzed by Pol III

primer removed by Pol I

DNA ligase seals remaining nicks

Summary of DNA replication in

prokaryotes

DNA synthesis is bidirectional

DNA synthesis is in the 5’ -> 3’ direction

the leading strand is formed continuously

the lagging strand is formed as a series of

Okazaki fragments which are later joined

DNA polymerases Five DNA polymerases have been found to exist in

E. coli

Pol I is involved in synthesis and repair

Pol II, IV, and V are for repair under unique conditions

Pol III is primarily responsible for new synthesis

Eukaryotic DNA replication

Not as understood as prokaryotic. Due in no

small part to higher level of complexity.

Cell growth and division divided into phases:

M, G1, S, and G2

DNA replication occurs during the S phase

RNA synthesis

Transcription

Template is DNA

Major enzyme: DNA directed RNA polymerase

No need for primers

5’ to 3’ direction

RNA synthesis

Requires a promoter region in the template DNA to which the RNA polymerse will bind

Promoter 40 base pairs upstream (-40) away from the start site (+1)

Three stages:initiation, elongation, termination

Termination may be rho factor dependent – rho factor terminates

synthesis

or rho factor independent – formation of a stable hairpin loop

Promoter 40 base pairs upstream (-40) away from the start site (+1)

INITIATION STEP

ELONGATION STEP

TERMINATION STEP

ρ-FACTOR INDEPENDENT- FORMATION OF HAIRPIN LOOP

Eukarotic transcription have 3 classes of

RNA polymerases

RNA pol I transcribes large ribosomal RNA

genes

RNA pol II transcribes protein encoding gene

RNA pol III transcribes small RNAs

(including tRNA and 5SRNA)

Post transcriptional modification of the

eukaryotic mRNA

Capping – methyl guanosine attachment at the

5’ end to protect the cleavage of the RNA by

exonucleases as RNA moves out of the nucleus

Addition of poly A at the 3’ end (200-250 long)

helps to stabilize the mRNA structure; increases

resistance to cellular nucleases

Splicing – removal of non coding sequences

(introns)

Protein synthesis

Translation

Based on the m-RNA sequence, genetic

code

Starts from 5’ end of the transcript

Occurs in the ribosomes

Activation of amino acids – attachment to the

tRNA

Initiation, elongation, termination

Genetic code

Triplet nucleotide – one amino acid

Nonoverlapping

No punctuation

Degenerate

Almost universal

Initiation

Initiation factors

Shine-Dalgarno sequence in mRNA

30S ribosome

N-formylmet

Inhibitors of protein synthesis

Postranslational modification

Protein folding –chaperones

Proteolytic cleavage (zymogens) – hydrolytic

enzymes in the gut

Amino acid modifications

Attachment of carbohydrates

Addition of prosthetic groups

Regulation of protein synthesis and gene

expression

20K to 25K genes in the human genome

Only a fraction of the genes are expressed at

any given time

Two types of gene expression: constitutive

and inducible

Inducible genes are highly regulated –

regulatory proteins, hormones and

metabolites