Gene structure, expression and regulation in prokaryotes

Gene structure, expression and regulation in prokaryotes

1. Bacterial genome

   • Circular chromosomal DNA
   • Typically a few million base pairs long
   • Most bacterial species contain a single type of chromosome
   • A few thousand different genes are interspersed throughout the chromosome, genes separated by short, intergenic regions
   • One origin of replication is required to initiate DNA replication
   • No nucleus
   • Haploid (one copy of every gene)
   • Small compact and repetitive

2. Plasmids

   • Replicate independently
   • Can integrate into chromosome
   • Not essential for survival
   • Fewer genes

3. Gene organisation in Prokaryotes

   • Organised into operons
     • A set of genes encoding proteins participating in the same metabolic pathway
   • Transcribed as polycistronic mRNA
     • Several proteins under influence of one promoter
       • Advantage: timing and amounts
       • Disadvantage: mutations inflexible

4. Transcription and translation

   • Occur at the same time in the “same cellular compartment”
   • Translation of an mRNA may begin before the mRNA is completely transcribed
   • Transcription unit
     • Promoter
       • Important in initiation
       • Conserved sequence of nucleotides in a DNA strand that is a binding site for RNA polymerase
       • Position and orientation of the promoter determines which strand is transcribed and where transcription starts
     • RNA coding region
     • Terminator
   • Transcription
     • Copying of template DNA strands
     • Strands grow in a 5' to 3' direction
     • RNA polymerases can initiate strand growth
     • DNA polymerases require a primer
     • Occurs in 3 phases
       • Initiation
         • Sigma factors recognise the promoter to initiate transcription
         • Promoter recognition Formation of transcription bubble Creation of bonds between rNTPs Escape from transcription apparatus from promoter
       • Elongation
       • Termination
         • Rho-independent Characterised by 1.) inverted repeats 2.) string of adenines -> slow down polymerase -> formation of hairpin structure followed by a string of uracils
         • Rho-dependent 1.) DNA region, that slows polymerase down 2.) Unstructured region -> binding site for rho protein, catches up, helicase activity
           • NO HAIRPIN STRUCTURE
   • Translation
     • Genetic code is read in triplicate but can be read in different frames
       • Genetic code and mutations
         • Silent mutation: the base change does not result in a protein change
         • Mis-sense mutation: a single amino acid is changed
         • Nonsense mutation: an amino acid codon is changed to a stop codon
         • Frameshift mutation: insertion or deletion of a number of nucleotides that is not a multiple of three
     • Different phases
       • Charging of tRNA
         • Amino acid links to tRNA
           • tRNA then binds its codon in RNA
             • Net result = amino acid is selected for by its codon
       • Initiation
         • Requires ribosomes and initiation factors
           • Complementary base-pairing between the Shine-Dalgarno sequence on the mRNA and the rRNA occurs
           • Small subunit binds to Shine-Dalgarnonsequence with the help of IF3
             • Then fMet-tRNA binds to initiation codon with the help of IF1, IF2 and GTP
               • This is called the 30s initiation complex
                 • Then IFs are removed and large subunit is added
                   • This is called the 70s initiation complex
                     • In summary the ribosome has been assembled on the mRNA and the first tRNA is attached to the initiation codon
       • Elongation
       • Termination
         • Once ribosome reaches stop codon RFs are recruted

5. Types of RNA found in prokaryotes

   • mRNA
   • tRNA
   • rRNA

6. Lac operon

   • lacZ
     • b-galactosiadase
       • Splits lactose into galactose and glucose
   • lacY
     • Lactose permease
       • Transports lactose into cell