dna and rna structurebiochem.vsmu.edu.ua/2_med_biochem_e/lecture_replication.pdf · dna and rna...
TRANSCRIPT
DNA and DNA and RNA RNA
structurestructure
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
9
N
N NH
NO
H2N
HN
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
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.→
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
• Chromatin – DNA plus different proteins• Histones – main proteins of chromatin
DNA in cells is a constituent of chromatin
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
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
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.
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
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
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
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’
GENETIC CODE - sequence of mononucleotides in mRNA that specifies the sequence of amino acids in peptide chain
CODON – mRNA triplet base sequence responsible for 1 amino acid
PROPERTIES OF GENETIC CODE
1. Unambiguous. In any organism each codon corresponds to only one amino acid.
2. Code is degenerate. There are multiple codons for most amino acids.
3. Universal. Codons are the same for all organism.
4. Without punctuation. There are no punctuations between trinucleotides.
5. Nonoverlapping. Codons do not overlap each other.
Structure of tRNAs
ANTICODON – triplet in tRNA that can complementary bind to codon of mRNA.
Such base pairing between codon and anticodon is responsible for the translation of genetic information from mRNA to protein.
R1 CH
NH2
COOH + HO P
O
O
OH
P
O
O
OH
P
O
OH
O Аденозин
R1 CH
NH2
CO P
O
O
OH
Аденозин + H4P2O7O
Aminoacyladenilate
RECOGNITION
Aminoacyl-tRNA-synthetase
Aminoacyladenilate + tRNA aminoacyl-tRNA + AMP
Activation of amino acids
Each amino acid has a specific tRNA
There is specific aminoacyl-tRNA-synthetase for each AA
Initiation of Translation•The translation complex is assembled at the beginning of the mRNA coding sequence
•Complex consists of: -Ribosomal subunits-mRNA template to be translated-Initiator tRNA molecule-Protein initiation factors
Initiator tRNA
•First codon translated is usually AUG
•The initiator tRNA recognizes initiation codons
-Bacteria: N-formylmethionyl-tRNA
-Eukaryotes: methionyl-tRNA
Elongation1) Positioning of the nextaminoacyl-tRNA in the A site 2) Formation of the peptide bound (enzyme – peptidyl transferase) between methionine and AA in A-centre. The residue of methionine is transferred on the amino group of another AA
3) Translocation – shift of ribosome by one codon. Methionyl-tRNA is released from P-centre. Dipeptidyl-tRNA moves from A-centre to P-centre.
Termination of Translation
• Ribosome comes to terminal codon UGA, UAG or UAA
• No tRNA molecules recognize these codons and protein synthesis stalls
• Protein termination factors F-1, RF-2, RF-3 split off synthesized polypeptide from the last tRNA
• Ribosomal complex dissociates
POSTTRANSLATIONAL MODIFICATION
1) Preparing of proteins for different functions
2) Direction of proteins to different locations (targeting)
1. Removing of methionine (formylmethionine)
2. Formation of disulfide and other bonds (secondary, tertiary structures)
3. Proteolytic cleavage
4. Modification of amino acid residues:
- Hydroxylation
- Glycosilation
- Phosphorilation
5. Joining of prosthetic groups or cofactors
6. Formation of the quaternary structure