A Molecular Biology primer….. Genetic information is carried on nucleic acids - DNA &RNA

The five bases are: adenine, guanine = purines

A Molecular Biology primer….. Genetic information is carried on nucleic acids - DNA &RNA The five bases are: adenine, guanine = purines and... thymine, cytosine, uracil = pyrimidines

A = T (or A = U) C G

Bases: adenine, guanine, cytosine, uracil, thymine Nucleosides: = base + sugar –on RNA sugar is ribose –adenosine, guanosine, cytidine, uridine, thymidine

on DNA sugar is deoxyribose deoxyadenosine, deoxyguanosine etc...

Nucleotides: base + sugar + phosphate RNA: adenylate, guanylate, cytidylate, uridylate

Nucleosides: base + sugar + phosphate RNA: adenylate, guanylate, cytidylate, uridylate DNA: deoxyadenylate, deoxyguanylate etc...

By convention, nucleic acid sequence refers to bases not nucleotides.

By convention, nuceic acid sequence refers to bases not nucleotides. Sequence is always written 5’ 3

Native DNA is normally double stranded

…the bases project inwards and are attracted by H-bonding.

Native DNA is normally double stranded …the bases project inwards and are attracted by H-bonding. RNA is normally single stranded.

At a particular temperature (T m ) H-bonds are broken and structure comes apart.

… = “melting” (or denaturation)

Cooling will lead to renaturation of structure.

Cooling will lead to renaturation of structure … = annealing.

Cooling will lead to renaturation of structure … = annealing. = basis in vitro of:hybridisation; PCR

Information flow is in one direction only: DNARNAProtein transcriptiontranslation

Information flow is in one direction only: DNARNAProtein transcriptiontranslation retroviruses

Information flow is in one direction only: DNARNAProtein transcriptiontranslation retroviruses Enzyme activity

Modifications: Cleavage: +

Modifications: Cleavage: + Ligation: +

Modifications: Cleavage: + (exo / endonucleases) Ligation: + (ligases) Splicing: +

DNA – two major functions:- Replication and transcription

DNA – two major functions:- Replication and transcription …in each case a template strand is needed.

In replication, the duplex splits locally and each strand acts as a template for synthesis of a new strand.

In transcription, the anti-sense strand acts as a template.

Therefore the RNA sequence is identical to the coding strand

RNA produced is the primary transcript. 1. 5’ capping with 7-methyl guanosine

RNA produced is the primary transcript. 1. 5’ capping with 7-methyl guanosine 2. 3’ cleavage to “poly A site”

RNA produced is the primary transcript. 1. 5’ capping with 7-methyl guanosine 2. 3’ cleavage to “poly A site” 3. polyadenylation (~250 A)

RNA produced is the primary transcript. 1. 5’ capping with 7-methyl guanosine 2. 3’ cleavage to “poly A site” 3. polyadenylation (~250 A) 4.Splicing of exons by excision of introns.

RNA produced is the primary transcript. 1. 5’ capping with 7-methyl guanosine 2. 3’ cleavage to “poly A site” 3. polyadenylation (~250 A) 4.Splicing of exons by excision of introns. 5.… = mRNA

RNA produced is the primary transcript. 1. 5’ capping with 7-methyl guanosine 2. 3’ cleavage to “poly A site” 3. polyadenylation (~250 A) 4.Splicing of exons by excision of introns. 5.… = mRNA = pre-mRNA Or heterogeneous nuclear RNAs (hnRNA)

Note that mRNA and pre-mRNA contain untranslated regions –3’ UTRs –5’ UTRs

Note that mRNA and pre-mRNA contain untranslated regions –3’ UTRs –5’ UTRs …these are “outside” the exon sequences.

Splicing is a variable process Dogma: 1 gene 1 polypeptide

Splicing is a variable process Dogma: 1 gene 1 polypeptide In fact: many products can be produced from each gene.

Splice variants: removal of selected exons.

Another reason….

Post-translational modification:

–Proteins –Glycoproteins –Phosphoproteins –lipoproteins

Post-translational modification: –Proteins –Glycoproteins –Phosphoproteins –Lipoproteins –The proteome is much more descriptive than the genome.

Translation There are 20 amino acids used in protein synthesis, but only 4 different bases.

Translation There are 20 amino acids used in protein synthesis, but only 4 different bases. …. So how does base sequence code for the amino acid sequence?

Each amino acid is coded for by 3 bases = a codon (a triplet of bases)

Each amino acid is coded for by 3 bases = a codon (a triplet of bases) Translation is co-linear

Each amino acid is coded for by 3 bases = a codon (a triplet of bases) Translation is co-linear … i.e. codon sequence matches the final  sequence.

–CACAACCAAUUU –his asn gln phe

–CACAACCAAUUU –his asn gln phe –“Start” and “stop” codons define the reading frame (coding region)

–CACAACCAAUUU –his asn gln phe –“Start” and “stop” codons define the reading frame (coding region) …. Which can be shifted - physiologically - by deletion / insertion mutations

ORF = open reading frame

= a suspected coding region once start / stop codons identified.