Computational Biology IComputational Biology ILSM5191LSM5191

Lecture 1: Introduction to Nucleic Acids – the Lecture 1: Introduction to Nucleic Acids – the building blocks of life.building blocks of life.

Aylwin Ng, D.Phil

2m of DNA,all 3 billion letters in the DNA code,

compacted into 46 chromosomes, and packed into a cell 0.0001cm across!

DNA & CHROMOSOMES

DNA densely packed into Chromosomes

Flow of Information in Living Systems

DNA RNA Protein

DNA SequenceSequence Implies StructureStructure Implies FunctionFunction

transcription translation

Central Dogma of molecular biology:

mRNA

mRNA Nascent polypeptide

ribosome

Transcription

Translation

Transport

functional protein

Post-transl. modif

Deoxyribonucleic acid (DNA) contains the information prescribing the amino acid sequence of proteins.

This information is arranged in units termed genes.

A GENE is the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide

Ribonucleic acid (RNA) serves in the cellular machinery that chooses and links amino acids in the correct sequence.

DNA and RNA are polymers of nucleotide subunits

NUCLEIC ACIDS

NUCLEOTIDE SUBUNITSNucleotide

Base

Phosphate group

Ribose orDeoxyribose(shown here)

• A nucleotide unit consists of a pentose sugar, a phosphate moiety (containing up to 3 phosphate groups) and a Base.

• Subunits are linked together by phosphodiester bond, to form a ‘sugar-phosphate backbone’:

NUCLEOTIDES• All nucleotides have a common structure

BASES

• 5 principal bases in nucleic acids:

A, G, C, T are present in DNA

A, G, C, U are present in RNA

NUCLEOSIDES & NUCLEOTIDES

ELUCIDATING THE STRUCTURE OF DNA

• James Watson (Cambridge University),

• Francis Crick (Cambridge University),

• Maurice Wilkins (King’s College London),

• Rosalind Franklin (King’s College London)- succeeded in obtaining superior X-ray diffraction data

Nobel Prize (Medicine) in 1962

X-RAY DIFFRACTION• Data showed that DNA has the form of a regular helix• Diameter 20 Å (2 nm)• Making a complete turn every 34 Å (3.4 nm)

i.e. 10 nucleotides per turn

BASE-PAIRING

Edwin Chargaff’s results (1952):Base compositions experimentally determined for a variety of organisms

Hydrogen bonding between complementary base pairs (A-T or G-C) holds the two strands together

DNA STRUCTURE• Native DNA (B-form) is a double helix of complementary anti-parallel chains.• Double helix is right-handed, with turns running clockwise along helical axis.

DNA REPLICATIONDNA REPLICATION

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”

-Watson & Crick, Nature (1953)

DNA REPLICATION• DNA replication is semi-conservative.

• IMPLICATION: the structure of DNA carries information needed to perpetuate its sequence .

• Demonstrated by Meselson-Stahl (1958)• Labeled parental DNA with ‘heavy’ density label by growing E. coli in medium containing isotope (e.g. 15N):

Light (14N)

Hybrid

Heavy (15N)

parental 1st Gen 2nd Gen

Both DNA and RNA chains are produced by copying of template DNA strands.

Nucleic acid strands grow in the 5’ 3’ direction.

Energetically unfavorable. Driven by energy available in the triphosphates.

DNA-dependent RNA polymerases can initiate strand growth but DNA polymerases require a primer strand.

NUCLEIC ACID SYNTHESIS

E. coli DNA polymerasesMain replicating enzyme

DNA repairDNA repair &

replication

DNA ReplicationClip

http://academy.d20.co.edu/kadets/lundberg/DNA_animations/DNAreplication.mov

BIDIRECTIONAL REPLICATION• DNA replication proceeds bidirectionally from a given starting site (Origin of

Replication), with both strands being copied at each fork.

Common features of Replication Origins (of E. coli, yeast, SV40)• Unique segments containing multiple short repeated sequences,

• Short repeated units recognised by multimeric proteins (which assembles DNA polymerases & replication enzymes),

• Origin regions contain an AT-rich stretch (less energy req.d to melt A.T base pairs).

Key events prior to the replication process (E. coli):

• Binding of DnaA protein at Origin separate (‘melt’) the strands.

• DnaC & DnaB bind at Origin.

• Then Helicase (DnaB) unwinding of duplex in opposite directions away from Origin.

• Unwinding of duplex is an ATP-dependent process.

• Single-strand binding (SSB) protein binds to the single-stranded (ss) DNA, preventing it from reforming the duplex state.

• Primases (RNA polymerase) bind to DnaB helicase primosome complex

• Primases dissociate after synthesizing short primer RNAs (complementary to both strands).

BIDIRECTIONAL REPLICATION

REPLICATION FORK

LAGGING-STRAND SYNTHESIS

TWO or just ONE Polymerase needed?

MAMMALIAN DNA POLYMERASESPriming

Main replicating

enzymeDNA repair

Mitochond. DNA

replication

REPLICATION IN EUKARYOTES• Very similar to replication in bacteria, differing only in details.

• DNA polymerase has primase activity generates RNA primers.

• DNA polymerase is the main replicating enzyme.

• Eukaryotic DNA polymerases appear to lack 5’ 3’ exonuclease activity needed to remove RNA primer from each Okazaki fragment.

• ‘Flap endonuclease’ (FEN1) initiates primer degradation by associating with DNA polymerase .

3’

3’

5’

5’

3’

5’Parent molecule

Chromosome end (Telomere)

Leading strand

Lagging strand

3’

5’

5’

3’

3’

5’

5’

3’

5’3’

5’3’

Molecule has become shorter

Missing Okazaki fragment

2 Daughter molecules

Next Generation (or Grand-Daughter) molecule

200bp200bp 200bp 200bp200bp

What happens at Telomeres?

The Solution: TELOMERASE• Telomeres can be extended by an independent mechanism.• Catalysed by TELOMERASE.• Enzyme consists of both protein & RNA.• RNA is 450 nucleotides long.

• Contains the seq. 5’-CUAACCCUAAC-3’ near its 5’ end.• Underlined seq. is the reverse complement of the human telomere repeat seq. 5’-

TTAGGG-3’.

• This allows telomerase to extend the 3’end a sufficient amount,

• to facilitate priming & synthesis of a new Okazaki fragment by DNA polymerase

generate a double-stranded end.

How TELOMERASE extends 3’-end