BIMM 100 Lecture 2

BIMM 100 Lecture 2 - BIMM100 Lecture 2 Key molecular...

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Unformatted text preview: BIMM100 Lecture 2 Key molecular processes I & II Reading: From 8/1/11 Lodish pages 111 ­125 8/2/11 Lodish pages 125 ­131 Read: Classic Experiment 4.1 – Deciphering the geneKc code (on Ted) Lecture summary •  TranscripKon overview –  “Big picture:” the 3 key types of RNA •  OrganizaKon: Bacteria vs. Eukaryotes •  The Enzyme: RNA polymerase •  The Process: 3 steps –  iniKaKon –> elongaKon –> terminaKon •  •  •  •  The Product: processing mRNA TranslaKon overview The Code Decoding –  tRNAs amino ­acyl synthetases •  Ribosomes and rRNA Key molecular processes I & II: transcripKon to translaKon Key: many transcripts funcKon as RNAs (tRNAs, rRNAs, miRNAs); they are never translated! nucleus nucleolus cytoplasm mRNA: coding, tRNAs: decoding, rRNAs: translaKng, miRNAs: regulaKon of target genes Next two classes: it’s all about RNA! •  mRNA –  Transcribed from DNA –  Processed in nucleus –  Decoded in cytoplasm •  tRNA –  –  –  –  –  Transcribed from DNA Processed in nucleus Structure Decoding Specificity •  rRNA –  –  –  –  Transcribed from DNA Processed in nucleus/nucleolus Structure FuncKon TranscripKon: an overview RibonucleoKdes (rNTPs) are polymerized in a chain (determined by base pairing) Phosphodiester bond formaKon is catalyzed by the enzyme RNA polymerase EnergeKcally favorable! The high energy bond between α and β phosphate is replaced by a phosphodiester bond between the nucleoKdes! AddiKonally, chain elongaKon is driven by pyrophosphatase, which cleaves the released PPi subunits into two molecules of inorganic phosphate RNA sequence is complementary to the template! A transcripKonal unit: the nomenclature Gene: region of DNA governing a funcKon. Product may be mRNA>protein or RNA itself Gene (includes regulatory sequences) Gene organizaKon in prokaryotes vs. eukaryotes •  Prokaryotes –  FuncKonal clustering of genes in operons •  Coordinate expression of all the genes in that operon (they are all transcribed and translated) –  TranslaKon of the mRNA actually begins while the RNA is sKll being transcribed! •  Eukaryotes –  No clustering of funcKonal genes! •  Each gene is transcribed from it’s own promoter –  Genes exist in pieces of exons (coding sequence) and introns (non ­coding sequence) which need to be further processed. •  5’ and 3’ modificaKons •  Splicing •  Transport Prokaryote gene organizaKon Prokaryotes ­ funcKonal clustering of genes in operons Simultaneous transcripKon and translaKon! EukaryoKc gene organizaKon is more complicated No clustering of funcKonal genes Primary transcript must be processed ­ cannot be directly translated! RNA polymerase Bacterial subunits: two large subunits (β & β’), two copies of a smaller subunit (α), and one stabilizing unit (ω). Eukaryotes and archaea are even more complex! “TranscripKon bubble” ­ the unwound DNA and acKve site! RNA polymerase mRNA: GREEN Template: RED RNA polymerase mRNA: GREEN Template: RED TranscripKon in acKon! TranscripKon (Step 1 ­ IniKaKon) TranscripKon (Step 2 ­ ElongaKon) TranscripKon (Step 3: TerminaKon) Complete ­ but not mature! mRNA processing (in eukaryotes) (m7Gppp) 5’ capping X Yeast Animals and plants (but not yeast) In primary transcript Contributes to stabilizaKon! Non ­templated addiKon DisKnguishing features of 5’ methylated cap? 1.  5’ ­5’ linkage of m7Gppp to iniKal nucleoKde 2.  Methyl group on 2’ hydroxyl of the ribose of Base 1 Only methylated in vertebrates! mRNA processing overview Gene Primary transcript capping Poly ­A addiKon (3’) polyadenylaKon Splicing (excision/ligaKon) Nuclear export Splicing (alternaKve splicing) provides choices •  Different exons may be removed in different cell types of the same organism •  Can yield different protein isoforms •  CriKcal: increases the repertoire or proteins that can be created from one gene! –  Nearly 60% of human genes are expressed as alternaKvely spliced mRNAs! There are more than 20 isoforms of this gene! Next two classes: it’s all about RNA! •  mRNA –  Transcribed from DNA –  Processed in nucleus –  Decoded in cytoplasm •  tRNA –  –  –  –  –  Transcribed from DNA Processed in nucleus Structure Decoding Specificity •  rRNA –  –  –  –  TRANSLATION! Transcribed from DNA Processed in nucleus/nucleolus Structure FuncKon The three roles of RNA: the process of translaKon 3. BUILDING (Ribosome, rRNA, and protein) Amino acid (aa) 2. DECODING (tRNA) 1.  CODING (mRNA) The code •  3 nucleoKdes = 1 codon •  GeneKc code is a triplet code –  20 “standard” amino acids •  43 = 64 possible codons –  61 specify amino acids, and three specify terminaKon (stop codons) •  Synonymous codons ­ different codon, same aa –  Except methionine and tryptophan •  Code is degenerate (there are redundancies) •  Code is nearly universal among different species (see table 4 ­2). Be comfortable “reading” these codes! (table 4 ­1) The code: how to read it! Reading frames (from start codon to stop codon) TranslaKon start and stop codons are not shown! Overlapping codes are possible (two different start codons)! Frameshiming can also occur (although this is rare) Two adaptors necessary for decoding 1.  tRNA 2.  Aminoacyl ­tRNA synthetases Two tRNA funcKons 1.  Must chemically link to a specific aa 2. Must base ­pair to an mRNA codon If errors occur in this process, then the wrong aa could be incorporated into a polypepKde chain! Luckily, synthetases have proof ­reading ability tRNA in two dimensions All tRNA fold into 4 base ­ paired stems and 3 loops 3’ CCA sequence also found in all tRNAs. Amino acid is anached at the 3’ A PosnranscripKonal modificaKons occur on some of the residues. Wobble pairing! •  Many cells contain < 61 tRNA genes –  How can that be possible? •  Wobble pairing: the capability of a single tRNA anKcodon to recognize more than one (but not every!) codon corresponding to a given amino acid •  Happens because of nonstandard pairing between bases in the “wobble” posiKon (the 3’ base in the mRNA codon and the 5’ base in the tRNA anKcodon). –  The 1st and 2nd bases of a codon almost always form standard Watson ­Crick base pairs, but four nonstandard interacKons can occur at the wobble posiKon Wobble pairing ParKcularly important: G ­U base pair fits almost as well as G ­C, so UUU and UUC are both read by tRNA with GAA anKcodon (phenyalanine) A is rare in the wobble posiKon. I (inosine; deaminated product of adenine found in plants and animals) is common ­ 4/6 leucine synonymous codons are read when I is in wobble posiKon (5’ ­GAI ­3’ anKcodon). Next two classes: it’s all about RNA! •  mRNA –  Transcribed from DNA –  Processed in nucleus –  Decoded in cytoplasm •  tRNA –  –  –  –  –  Transcribed from DNA Processed in nucleus Structure Decoding Specificity •  rRNA –  –  –  –  Transcribed from DNA Processed in nucleus/nucleolus Structure FuncKon MORE TRANSLATION! rRNAs are essenKal components of ribosomes RNA structural complexity in the bacterial 16S rRNA molecule Structural role: acts as scaffold to define posiKon of the ribosomal protein. 3’ end contains Shine ­Delgarno sequence (binds upstream to the AUG start on mRNA). Interacts with 23S, which aids in the binding of the two subunits (the 50S and 30S). pink= conserved structural elements The protein and RNA E.Coli 70S ribosome E, P, and A sites line the interface of the 50S and 30S subunits rRNA is light purple (and light green), while the protein components are colored darker. The 5S rRNA component is colored dark blue. Note that rRNAs line the inside inacKon areas (E, P, A), while most of the proteins are on the outside of the complex. Lecture summary •  TranscripKon overview –  “Big picture:” the 3 key types of RNA •  OrganizaKon: Bacteria vs. Eukaryotes •  The Enzyme: RNA polymerase •  The Process: 3 steps –  iniKaKon –> elongaKon –> terminaKon •  •  •  •  The Product: processing mRNA TranslaKon overview The Code Decoding –  tRNAs amino ­acyl synthetases •  Ribosomes and rRNA Key molecular processes I & II: transcripKon to translaKon Next lecture: TranslaKon & DNA replicaKon nucleus nucleolus cytoplasm mRNA: coding, tRNAs: decoding, rRNAs: translaKng ...
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