Welcome toIntroduction to BioinformaticsFriday, 1 September Introduction to Molecular Biology DNA to protein
Transcription Translation Replication The “Central Dogma” of Molecular Biology Term coined by Francis Crick in 1956 to describe the flow of information in the cell DNA RNA Protein
What is the nature of the Gene? Oswald Avery, Colin MacLeod & Maclyn McCarty first show that DNA is the “genetic principle” Enzymes used to degrade proteins
What is the structure of DNA? 1952 Rosalind Franklin and Maurice Wilkins produce X-ray diffraction images of DNA crystals that suggested that DNA must have some helical arrangement
What is the structure of DNA? 1953 Francis Crick and James Watson put together all of the clues and correctly deduce that DNA is a Double Helix
DNA base pairing occurs through hydrogen bonds A:T pairs: 2 bonds G:C pairs: 2 bonds
The double helix strongly suggested that DNA replication might proceed by a “semiconservative” process
Genes control the amino acid sequence of proteins • 1957 – Vernon Ingram shows that sickle cell haemoglobin varies from wild type by the substitution of one amino acid
Genes control the amino acid sequence of proteins Alteration of amino acid sequence is also observed in all other hereditary anaemias!
DNA cannot directly specify the sequence of amino acids in proteins • Protein synthesis in eukaryotic cells known to take place in the cytoplasm • There must therefore be a SECOND information containing molecule that gets its specificity from DNA, but then moves to the cytoplasm • Attention immediately focuses on RNA – was easy to imagine that it could be produced from a DNA template • Torborn Caspersson and Jean Brachet demonstrated that RNA was mostly in the cytoplasm Jean Brachet (1909-1998)
The case for RNA Missing methyl group in uracil relative to thymine Hydroxyl group Chemically very similar to DNA
RNA Polymerase is a molecular machine that carries out transcription
RNA is synthesised in the nucleus but travels to the cytoplasm Cells pulse-labelled with 3H coupled cytidine T = 15 minutes T = ~90 minutes D.M. Prescott
Ribosomes are the site of protein synthesis ribosomes studding the endoplastic reticulum Shown using radio labelled amino acids in conjunction with ultracentrifugation to isolate Different cell fractions. Where does the radioactivity end up at various times?
Ribosomesand associated rRNAs are the factories for protein synthesis More on ribosomes in BNFO 507!
Nature of the genetic code • Obvious early on most likely a triplet code in order to code 20 amino acids: • 4 x 4 nucleotides can specify 42 = 16 amino acids • 4 x 4 x 4 nucleotides can specify 43 = 64 amino acids • Code must be redundant • Not overlapping – Sydney Brenner’s thought experiment • Marshall Nirenberg and Heinrich Matthaei showed that a homopolymer (UUUUUU…. etc. ) produced a poly-phenylalanine protein
Khorana's synthetic RNA approach to cracking the genetic code • Example RNA with two repeating units • RNAs with two repeating units: • (UCUCUCU → UCU CUC UCU) produced a polypetide consisting of alternating Serine and CUC codes for Leucine • RNAs with three repeating units: • (UACUACUA → UAC UAC UAC, or ACU ACU ACU, or CUA CUA CUA) produced three different strings of amino acids • RNAs with four repeating units including UAG, UAA, or UGA, produced only dipiptides and tripeptides thus revealing that UAG, UAA and UGA are stop codons.
Crick’s adaptor hypothesis • Can folded RNA act as the template for protein synthesis? • Seems unlikely: • the nucleosides chemically want to react with water soluble groups • but many amino acids are polar • no clear way to discriminate chemically similar amino acids Crick proposes that an adaptor molecule must fit between RNA and the incoming amino acids, but its nature is unknown Incoming amino acid Adaptor molecule RNA
Translation proceeds through a tRNA intermediate
The genetic code is (almost) universal
AAAAAA...AAA Splicing AAAAAA...AAA Study Question 8Why do introns exist? Splice boundaries highly conserved
Protein #2 DNA-bindingprotein Study Question 8Why do introns exist? Protein #1 hormoneresponsiveness protein kinase DNA binding chromosomal rearrangement Hormone-responsive protein kinase
Study Question 8Why do introns exist? hormoneresponsiveness protein kinase DNA binding AAAAAA...AAA New protein:Hormone-responsiveDNA-binding protein
Study Question 11Degeneracy and frequency of amino acids Most commonLeu Gly Ser Least commonTrp Met His
Study Question 12Single mutation from AGA Silent: | Hydrophilic/Hydrophilic: |
Study Question 12Single mutation from AGA Silent: | Conservative: | Hydrophilic/Hydrophilic: | | Hydrophilic/Hydrophobic: |
Study Question 12Single mutation from AGA Silent: | | Conservative: | Hydrophilic/Hydrophilic: | | | Hydrophilic/Hydrophobic: | Other: |
Enzymes lower the activation energies associated with biochemical reactions DG Typical energy of activation is 20-30 kcal/mol
Eukaryotic mRNA must often must be spliced in order to produce a mature transcript Exons often correspond to functional protein domains and alternative splicing can give rise to variant proteins