Molecular mechanisms of DNA replication. Transcription. The painting “Dawn of the Double Helix”...

Molecular Molecular mechanisms of mechanisms of DNA replication. DNA replication. Transcription.Transcription.

The painting “Dawn of the Double Helix” composes the

DNA duplex as human figures. The theme in this painting is

“Life forms: The basic structures that make our

existence possible”.

• Two types of nucleic acids – DNA and RNA

• Genome - the genetic information of an organism. The genomes of all cells are composed of DNA.

• Nucleic acids are biopolymers consisting of nucleotides

• Nucleotides have three components: (1) A weakly basic nitrogen base (2) A five-carbon sugar (3) Phosphate

Ribose and Deoxyribose

Ribose is the constituent of RNA

Deoxyribose is the constituent of DNA

Рибоза

Дезоксірибоза

Nitrogen Bases of Nucleic Acids

N

N NH

N

NH2

1

2

3

65

4

7

8

9N

N NH

NO

H2N

H

N

NH

O

O

H2

3

4

1

5

6

N

NH

NH2

O

HN

NH

O

O

CH3

The nitrogen bases are derivatives of either pyrimidine or purine.

Adenine CytosineGuanine Thymine Uracil

NucleosidesNucleosides are composed of ribose or deoxyribose and a heterocyclic base.

Structure of mononucleotide

HH

OH OH

H H

O

N

N N

N

NH2

CH2OP

OH

OH

O

Adenosine mononucleotide

O

5'

CH 2 A.O.

OH

O n

3'

3'

O

PO O

OH

P

O

OH

O P

O

OH

OH

CH 2

OH

O

5' A.O.n+1

..

O

5'

CH 2 A.O.

O

O n

3'

PHO O

OH

OA.O.

n+1

+ ФФн

O

CH 2

Formation of DNA chain (5’-3’ direction)

Nucleotides joined by 3’-5’ phosphodi-ester linkages

One end of the polynucleotide chain is said to be 5’ (no residues are attached to 5’-carbon) and other is said to be 3’. Direction from the top to bottom is called 5’→3’, from bottom to top – 3’→5.

Primary structure of nucleic acids

Two Antiparallel Strands Form a Double Helix

• Bases in opposite strands pair by complementary hydrogen bonding

• Adenine (A) - Thymine (T)

• Guanine (G) - Cytosine (C)

DNA is Double-Stranded

Watson JamesCrick Francis

Two strands run in opposite directions

The double helix of DNA was discovered in 1953 by Crick F. and Watson J. Nobel prize in 1962.

Chemical structure of double-stranded DNA. The two strands run in opposite directions. Adenine in one strand pairs with thymine in the opposite strand, and guanine pairs with cytosine

• Comple-mentary base pairing and stacking in DNA

Two-stranded struc-ture of DNA

•Chromatin – DNA plus different proteins

•Histones – main proteins of chromatin

DNA in cells is a constituent of chromatin

Chromatin structure

•DNA is packaged by coiling of the into a solenoid (helix) structure

Types of RNA

(1) Transfer RNA (tRNA)•Carries amino acids to translation

machinery•Very stable molecules

(2) Ribosomal RNA (rRNA) •Makes up much of the ribosome•Very stable, majority of cellular RNA

(3) Messenger RNA (mRNA) •Encodes message from DNA to

ribosomes•Rapidly degraded by nucleases

DNA ReplicationDNA Replication

The flow of genetic information in a typical cell

The main postulate of molecular

biology

DNA

RNA

protein

Semiconservative mechanism of DNA replicationThe two strands separate, and each strand is copied to generate a comple-mentary strand. Each parental strand remains associated with its newly synthesized complement, so each DNA duplex contains one parental strand and one new strand.

Replication – synthesis of DNA on the DNA template

A model for DNA replication

Components which are necessary for replication

•Enzymes (most important – DNA-dependent DNA polymerase)

•Protein factors

•Parental DNA

•ATP, GTP, ТТP, CТP

•Ions Mg і Zn

Helicase

Primase

Okazaki fragmentsPrimer

Leading strand

Lagging strand

5’

5’

5’

3’

3’

3’

DNA replication• In eukaryotes

the replication begins in many points simultaneously

• V-shape – replication forks - the point of the beginning of replication

• Helicase – enzyme untwisting the double strand

•Replisome - protein machinery for replication

•Replisome contains: primosome, DNA polymerase III, proteins

•Helicase is a constituent of primosome

•Bidirectional DNA replication in E. coli

•New strands of DNA are synthesized at the two replication forks where replisomes are located

DNA polymerase•DNA polymerase III – the main enzyme of

replication responsible for the chain elongation

•Appropriate nucleotides are inserted in the correct positions according to the complementary principle

•DNA polymerases only synthesize new strand in the 5’-3’ direction.

• Напрямок синтезу 5’-3’, антипаралельно до матричного ланцюга

DNA polymerase synthesizes two strands simultaneously

Because DNA polymerases only polymerize nucleotides 5 ’3’, both strands must be synthesized in the 5’3’ direction. Thus, the copy of the parental 3’5’ strand is synthesized continuously; this newly made strand is designated the leading strand. As the helix unwinds, the other parental strand (the 5’3’, strand) is copied in a discontinuous fashion through synthesis of a series of fragments - Okazaki fragments; the strand constructed from the Okazaki fragments is called the lagging strands

Синтез відстаючого ланцюга відбувається

дискретно• Lagging strand

is copied in a discontinuous fashion (Okazaki fragments)

• The formation of a phosphodi-ester linkage between of adjacent Okazaki fragments is catalized by ligase

Okazaki Model

Reiji Okazaki provided experimental evidence for

discontinuous DNA synthesis

RNA Primer Begins Each Okazaki Fragment

•Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork - primer

•DNA pol III uses the RNA primer to start the lagging-strand DNA synthesis

•Replisome - includes primosome, DNA pol III

•Each Okazaki fragment has the primer

Okazaki Fragments Are Joined by Action of DNA Polymerase I and

DNA Ligase

•DNA pol I removes the RNA primer at the beginning of each Okazaki fragment

•Synthesizes DNA in place of RNA

DNA polymerase I activities

DNA ligase

•Catalyzes the formation of a phosphodiester linkage between of adjacent Okazaki fragments

Repair of Damaged DNA

•DNA is the only cellular macromolecule that can be repaired

•DNA damage includes: -base modifications-nucleotide deletions or

insertions-cross-linking of DNA strands-breakage of phosphodiester

backbone

•Reparation – enzymatic deletion and synthesis of the damaged DNA fragments

•Recombination - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosome

•Transposition – dislocation of gene or group of genes from one place to another

ТРАНСКРИПЦІЯТРАНСКРИПЦІЯ

NECESSARY COMPONENTS

•DNA matrix

•DNA-dependent RNA-polymerase

•АТP, GТP, CТP, UТP

•Мg ions

DIFFERENCE FROM REPLICATION

•Only one strand is used as a matrix

•Only the part of DNA is transcribed (not the entire chain)

RNA Polymerase•There are 3 RNA-polymerases in eukaryotes (for mRNA, rRNA, tRNA)

•RNA pol is core of a larger transcription complex

•Complex assembles at one end of a gene (promoter) when transcription is initiated – transcription initiation

•DNA is continuously unwound as RNA pol catalyzes a processive elongation of RNA chain

The Chain Elongation Reaction

•Mechanism almost identical to that for DNA polymerase

•Growing RNA chain is base-paired to DNA template strand

•Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template

•New phosphodiester bond formed

• Direction 5’-3’

• Speed - 30-85 nucleotides/sec

Initiation and elongationsteps of transcription

The transcription bubble

•RNA poly-merase reaction

•RNA poly-merase reaction

Transcription Termination

•Only certain regions of DNA are transcribed

•Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences

PROCESSING

•Transcription occurs in the nucleus, translation in the cytoplasm

•Eukaryotic mRNA is processed in the nucleus

•In some mRNA, pieces are removed from the middle and the ends joined (splicing)

•Introns - internal sequences that are removed from the primary RNA transcript

•Exons - sequences that are present in the primary transcript and the mature mRNA

•Specific enzymes cut out introns and join exons - splicing

introns

Primary transcript

mRNA

transcription

splicing

DNA

exons exones

PROCESSING

7-methylguanosine (CAP)

Poly-A (TAIL)

5’ 3’