a molecular biology primer….. genetic information is carried on nucleic acids - dna &rna

• A Molecular Biology primer…..

• Genetic information is carried on nucleic acids - DNA &RNA

• 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

• 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 (Tm) H-bonds are broken and structure comes apart.

• At a particular temperature (Tm) 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:

DNA RNA Proteintranscription translation

Information flow is in one direction only:

DNA RNA Proteintranscription translation

retroviruses

Information flow is in one direction only:

DNA RNA Proteintranscription translation

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.

• 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.

• Splice variants: removal of selected exons.

• Another reason….

• Another reason….

• Post-translational modification:

• 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

ORF = open reading frame

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