molecular genetics

what are the sites on ribosomes

• P - site (peptidyl site) - binds to the tRNA holding the growing polypeptide chain of amino acids

• A - site (acceptor) - binds to tRNA holding the new amino acid to be added to the polypeptide chain

• E -site (exit) - binds a tRNA that already unloaded its amino acid and it is going to be released

how does termination take place?

• a release factor binds directly to the stop codon in the A site

• addition of H20 molecule instead of an amino acid

what are polyribosomes?

• in prokaryotes mRNA is translated to protein as soon as its made

• in eukaryotes mRNA has to be transported out of the nucleus before it can be translated

• in both cases several ribosomes can simultaneously translate one mRNA molecule

what are open reading frames?

• contigous sequence of DNA or RNA that starts with a start codon and ends with stop codon indicating a region with potential of being translated into a protein

• does not include introns

• dna sequences read in 5’ to 3’ direction

operons in bacteria

gene expression in eukaryotes

• control expression of individual genes

scales of regulation:

• epigenetic

• transcriptional

• post - transcriptional

• translationl

• post - translational

what is gene expression?

• the overall process of producing a protein from the gene that encodes the protein

• Promoter activation
  • Initiation of transcription and translation
  • Alternative splicing
  • Translation
  • Protein folding and targeting

how can mRNA be measured using the nothern blot

• transcriptome = entire set of RNA molecules (all mRNA)

1. the northern blot:

• rna gel electrophoresis - smaller mRNA molecules travel furtherst

• rna with gel is blotted onto a membrane that binds to RNA

• short piece of single stranded DNA complementary to RNA is incubated with the membrane

• the dna probe only anneals to the mRNA of interest on the membrane - the dna probe is fluorescent or radioactive

• the more abundant the mRNA the more probe sticks to it

• the amount of probe is measured imaging equipment

How can mRNA be measured using RT - PCR

• RT - retrotranscription

• retrotranscriptase - viral enzyme that synthesises ssDNA from mRNA

• polyTprimer binds to the mRNA poliA tail

• RT synthesises cDNA of all the genes in the tube

• we select gene of interest with primers and PCR

what are microarrays?

• microarrays - allows the study of whole transcriptomes

• each well/cell in the grid contains unique short RNA or DNA probes complementary to genes of interest

• array is incubated with mRNA or cDNA of a sample

• Fluorescent labelling allows detection of the amount of nucleic acid bound to each short DNA sequence on the array

• spot intensity provides mRNA quantity for each single mRNA

how else can you study whole transcriptomes

• RNA sequencing

• next generation sequencing allows whole genomes to be sequenced relatively cheaply

• applied to sequence all mRNA’s from an organism

• the number of times a sequence is obtained for the mRNA from each gene reflects the mRNA expression level

• rna sequencing is inexpensive and large number of sample can be processed together

• can be used with organisms having no known genome sequence

How to measure proteins

• measuring DNA/RNA is easy due to complementary base pairing

• protein detection requires production of an antibody that recognises the protein

• relies on ability of mammals to produce an antibody to foreign protein

western blotting:

• mix of proteins extracted from an organisms is separated using polyacrylamide gel electrophoresis (PAGE)

• PAGE gels are thin and normally arranged vertically

• the protein extract is treated with detergent that gives it negative charge

• darker bands indicate more antibody has attached so more of the protein of interest is present

what are small scale mutations

substitutions:

• silent

• missense

• nonsense

• all point mutations

insertions or deletions:

• frameshift missense

• frameshift nonsense

• gain/loss of amino acids

what are large scale mutations?

• alterations of chromosome numbers

• alteration of chromosome structure:

• deletion

• duplication

• inversion

• translocation

what is thalassaemias

• mutation which affects production of the a and b chains of haemoglobin leading to abnormal ratios

• α-thalassaemia: synthesis of the α chain absent or reduced –
  usually as a result of deletion of one or more α-globin genes

• β-thalassaemia: synthesis of β chain absent or reduced –
  usually as a result of defective processing of β-globin mRNA