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Lecture 4

BIOL 130 Lecture 4: Unit 5 Gene Expression DNA to RNA

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BIOL 130
Heidi Engelhardt

Unit 5 Gene Expression DNA to RNA • Central Dogma of (Molecular) biology DNA – RNA – Protein • Genotype – Effect/ activity of protein (proteome) (change the protein content)– phenotype Recap – RNA vs. DNA • Monomers themselves – Deoxy and ribo • 4 nucleotides – thymine in DNA and Uracil in RNA DNA is transcribed by RNA polymerase • Nucleotides in both include sugar, phosphate and base 3D structure of DNA vs. RNA • DNA – antiparallel • RNA- in the same strand 3D structure Modifications to central dogma • many genes code for RNA molecules that are not mRNA Prokaryotic RNA Polymerase (RNApol) o do not code for proteins • large, globular enzyme o these RNAs are involved in: with several channels o regulation of gene transcription running through it o processing of mRNA prior to translation o Recognizes start o translation point ▪ transport of amino acids o Helps in physically ▪ catalyze the formation of peptide bond separating the DNA Diff types of RNA found in the cell • active site is at the intersection of these channels • holoenzyme made up of a core enzyme, which has the ability to synthesize RNA and a regulatory subunit (sigma factor) Sigma Factor recognizes a promoter sequence • most bacteria have several types of sigma proteins • e.g. E coli has 7 types of sigma proteins Transcription from a DNA template • each sigma binds to • Only one strand of DNA transcribed o template strand promoters with slightly • The other DNA strand called non-template (coding) different sequences • sigma factors recognizes the -10 and -35 boxes strand o sequence of ‘coding’ DNA strand matches RNA, Prokaryotic promoters and inhibition of transcription except that RNA has uracil (U) in place of thymine (T) • transcription initiated at specific sections of DNA called promoters • always adding to the 3’ end of the strand o regions on non-template strand, 40-50 bp long • promoters have two key regions: Formation of phosphodiester bonds • Enzyme – RNA polymerase mediates the bond – o 10 box – 10 bases upstream from start site o 35 box – 35 bases upstream catalyzes the reaction (transcription starts at +1) ▪ typical sequences found at boxes, rest of promoter highly variable • transcription begins when sigma identifies and binds to –10 and –35 boxes, properly orienting the RNA polymerase holoenzyme for transcription at start site Which strand is used as the template • promoters are asymmetric  binds polymerase in only one direction • strand can only be built 5’ to 3’ • • strand tht has the promoter is the non-template strand Transcription in Bacteria: Termination • which strand? • RNApol reaches a transcription termination signal o  depends on gene in DNA template o codes for RNA that folds back on itself (short double helix), forming hairpin structure o hairpin disrupts transcription complex ▪ RNApol separates from RNA transcript ▪ As soon as the hairpin loop forms – transcription stops Higher order of DNA Structure • DNA molecules can adopt higher order structure o Allows for compact packaging Eukaryotic RNA polymerase • RNA polymerase – messenger RNA Recap of transcription in Prokaryotes • Transcription in Eukaryotes vs. prokaryotes • TBP (TATA binding protein) • splicing carried out by spliceosomes recognizes promoter o consist of 5 small nuclear ribonucleic particles sequence (RNA-protein complex) (snRNPs, pronounced • TBP + TFIID distorts helix, ‘snurps’) RNA + >100 proteins allows other factors (TFIIA, B, ▪ catalytic activity provided by RNA C, etc) to pile on to form component ‘transcription initiation • ‘ribozymes’ complex’ Introns are spliced out TATA Binding Protein • branch point A ‘attacks’ 5’ splice • subunit of TFIID that site  cuts sugar-phosphate recognizes and binds to backbone TATA box within promoter • cut end forms covalent bond with o causes kinks and partial ribose sugar group unwinding of double • lariat structure eventually helix degraded o modifications - How many exons in a typical eukaryotic gene? • Highly variable Organization of Prokaryotic Vs. Eukaryotic genes • coding sequences of eukaryotic genes (exons) Disadvantages of RNA splicing interrupted by non-coding sequences (introns) • more steps  more work! • more steps  more opportunity for error o mutations of splice sites can result in ▪ loss of exons, inclusion of introns ▪ shift in location of splice Advantages of RNA Splicing • can create different proteins from same gene / same primary mRNA transcript depending on cell type, stage development, gender, etc. Eukaryotic DNA: Coding & non-coding Regions ‘splice variants’ • to make functional mRNA, entire gene (introns + exons) transcribed (primary transcript) • after capping, while still being transcribed, RNA splicing (removal of introns) begins o all AAs except methionine (Met) and tryptophan (Trp) specified by more than one codon o but one codon never specifies more than one AA • code is almost universal • α- tropomyosin interacts with actin, expressed in all cell types o different isoforms have different functions ▪ muscle cells  regulation of contraction Are there any exceptions to the genetic code? • same codons assigned to the same amino acids and ▪ other cell types  involved in microfilament functions to the same START and STOP signals in the vast majority of genes in animals, plants, and microbes Only mature mRNA is exported from nucleus BUT some exceptions have been found: • cap and poly-A tail are ‘marked’ by proteins • cap must exist to leave nucleus through nuclear • mostly assigning some of the three STOP codons to pores
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