BIOL 310 - Ch 14 Lecture Notes

eukaryotic gene

• TATA box w/promoter

• start codon

• introns & exons

• poly A consensus sequence & poly A tail

• stop codon

mature messenger RNA

• contains 5’ untranslated region (UTR), protein coding 3’ region, untranslated region

• prokaryotes have the Shine-Dalgarno sequence AGGAGG

  • contains the start codon

• eukaryotes have the Kozak sequence (gcc)gccRccAUGG

  • also contains the start codon

Post-transcriptional modifications

• modifications made on the mRNA transcript for it to become mature

  

• addition of a 5’ cap, cleave the 3’ end, RNA splice, RNA editing (alter sequence of RNA)

• 5’ cap

  

  • protects the 5’ end since it is exposed to cellular surroundings and it is at risk for denaturing

    • exonucleases can break it down

  • 7-methylguanine added since there is no enzyme that readily complexes to it and can take it apart

• 3’ Poly-A tail

  • to prevent degradation by exonucleases

  • length of tail & longevity of molecule

  • necessary for mRNA transport out of nucleus (for prokaryotes)

  • consensus sequence transcribed; recruits enzyme to cleave RNA & add a string of A’s

  • polydenylatecyclase is the enzyme responsible for adding the poly a tail

    • does this without a template — adenine is the only base that can be added without a template

RNA splicing

• eukaryotes only

• introns = interfere | exons = expressed

• start with exon and end with an exon

• alternation of intron and exon

  • there is always one fewer introns than exons

• consensus sequences at the border of exons & introns

  • complex of proteins to recognize the cite

• 5’ splice site splits exon 1 from the intron and exon 2 to yield the Lariat

  • the 5’ end of the intron then loops back onto itself to form a phosphate bond with an adenine

  • cut 3’ end of exon 2

  • join 3’ end of exon 1 with 5’ end of exon 2

  • the lariat is mature and ready for translation

• spliceosome - composed of proteins & DNA

  • very organized process since could end up with a frame shift (insertions or deletions)

  • U1 +U2

    • U1 binds to exon intron border in a sequence specific manner

    • U2 binds to the branch point (the adenine phosphate bond)

  • U4 + U5 + U6

    • U4 U5 U6 complex together and interact with U1 that cause the formation on the branch point and the breaking of the exon intron phosphodiester bond

    • the intron exon 2 border cite gets cleaved & the 3’ end of exon 1 joins with the 5’ end of exon 2

    • this is an energy neutral process — powered by proton shuttle

• self-splicing introns (no energy required)

  • introns forming complex structure where 3’ and 5’ are close → nucleophilic attack which leads to two phosphodiester bonds being broken and one created

    • exon 1 and exon 2 are brought together regardless of whether it is group I (internal) or group II (external)

  • both are considered self-splicing because of this ability to place the two sites next to each & break bonds

• alternative splicing - in eukaryotes ONLY!!

  • a gene with multiple exons does not dictate whether all the exons must end up in the mature mRNA

  • first exon has start codon & last codon has stop codon

  • it is possible to have different outcomes (end product - after splicing)

    • all exons can be present

    • just exon 1 & exon 3 are present

    • just one exon

    • alternate splice sites that lead to just one exon present

  

  • gives the genome a high degree of expression capacity

    • 75,000 genes are alternative splicing products

    • exons will be in increasing order (no shuffling of exons)

editing with guide RNA

• take pre-edited mRNA & hybridize it guide RNA to yield a complementary strand

• the guide RNA has folds in it and stretches — there will be breaks wherever there are folds which are filled in by RNA dependent RNA polymerase

  • this results in mature RNA that has a different coding sequence compared to the original pre-edited mRNA

mRNA process overview

• lots of collinearity

• gene ends up in RNA & protein

other RNAs

• tRNA — transfer RNA

  • RNA molecule that functions as an RNA molecule that folds on itself to make an acceptor arm for the amino acid

  • has an anticodon arm that will interact with the ribosome

• rRNA — ribosomal RNA

• micro RNA (miRNA) & small interfering RNA (siRNA)

  • involved in RNA interference and gene silencing

• long non-coding RNA (IncRNA)

  • >200 bp

  • >10,000 in human genome

  • function is unknown

    • some control gene expression for cell cycle regulation, chromosome stability, chromatin structure

      • Xist — X chromosome to barr body & regulates dosage compensation