BIMM 100 Lecture 3 with questions

BIMM 100 Lecture 3 with questions - BIMM100 Lecture 3...

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Unformatted text preview: BIMM100 Lecture 3 Key molecular processes II & III Sec8ons start today! Reading: From 8/2/11 Lodish pages 125 ­131 8/3/11 Lodish 132 ­145 Watch: hIp://www.youtube.com/watch?v=u9dhO0iCLww a truly epic movie on protein synthesis & hIp://www.youtube.com/watch?v=d1UPf7lXeO8 a brilliant DNA rap Key molecular processes II & III: transla8on, DNA replica8on, and repair nucleus nucleolus cytoplasm mRNA: coding, tRNAs: decoding, rRNAs: transla8ng Some issues… •  iClicker ques8on from yesterday ­ ques8ons and discussion •  Wobble pairing ­lots of ques8ons! •  iClicker registra8on! –  If your serial number has been rubbed off, you need to come see me ­ I can “read” what your serial number is on my base sta8on. –  S8ll keep vo8ng, even if your serial number is not registered. I can do that step later, manually (I just don’t want to do 200 of them manually…) Clicker Ques8on For each tRNA, there may be Your answers in class: A) more than one amino acid B) only one amino acid C) more than one codon D) only one codon E) no available codons KEY: The gene8c code is degenerate but unambiguous. This means that each codon codes for a single amino acid, and each tRNA carries only one amino acid, but there can be more than one codon that specifies a par8cular amino acid. Clicker Ques8on ­ CLARIFICATION! For each tRNA, there may be A) more than one amino acid ­ B) only one amino acid C) more than one codon D) only one codon E) no available codons NO! each tRNA recognizes a specific aa. This specificity is imparted by the aminoacyl ­tRNA synthetase that recognizes a specific aa, linking it to a specific tRNA via a high energy ester bond. There is one excep8on ­ MetRS (an aminoacyl ­tRNA synthetase) recognizes and charges tRNAMet and tRNAiMet. However, only tRNAiMet can bind at the P site to begin polypep8de synthesis. YES ­ see above YES ­ each tRNA can recognize more than one codon. WOBBLE PAIRING! NO ­ each tRNA can pair with more than one codon ­ this explains how there can be more codons than tRNAs! This answer is completely wrong. Thankfully, no one picked it. Good job! Brief recap •  One tRNA molecule binds to a specific amino acid. •  Each amino acid has a subset of tRNAs that code for it (i.e. each amino acid can aIach to more than one tRNA). –  Example: Proline (an amino acid) is aIached to tRNAs with the an8codon sequences of: 5’ ­AGG ­3’, GGG, UGG, CGG. •  tRNAs can pair with more than one codon (because of wobble base pairing!). So, let’s review wobble base pairing. Lecture summary •  Finishing transla8on –  tRNA structure and func8on –  Wobble basepairing –  rRNA and ribosomes –  The process: 3 steps •  ini8a8on –> elonga8on –> termina8on •  Inhibi8ng the process: toxins and pharmaceu8cals •  DNA replica8on –  Overview –  Ini8a8on –  Bi ­direc8onal synthesis and polarity –  Proofreading and repair More 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 Func8on Brief review The three roles of RNA: the process of transla8on 3. BUILDING (Ribosome, rRNA, and protein) Amino acid (aa) 2. DECODING (tRNA) 1.  CODING (mRNA) tRNA in two dimensions Yeast tRNAala All tRNA fold into 4 base ­ paired stems and 3 loops 3’ CCA sequence also found in all tRNAs. Amino acid is aIached at the 3’ A PosIranscrip8onal modifica8ons occur on some of the residues. 30 ­40 tRNA genes in bacteria > 50 ­100 in animals and plants If we have fewer than 61 tRNA genes, how do we read the code? Wobble pairing Par8cularly important: G ­U base pair fits almost as well as G ­C, so UUU and UUC are both read by tRNA with GAA an8codon (phenyalanine) A is rare in the wobble posi8on. 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 posi8on (5’ ­GAI ­3’ an8codon). Wobble pairing ­ more detailed explana8on! KEY: the first two bases of the mRNA codon bind 8ghtly (Watson ­Crick base ­pairing) to their complementary bases in the tRNA molecule. The third base can either pair with its “normal” partner (i.e. A with U or C with G), or it can pair with different bases. Why? It’s a structural issue ­ if the first two bases are 8ghtly bound, the third base is not as important. Reading this figure: if tRNA has C in wobble posi8on, then it will pair with a G in the mRNA only. If it has an A, it will pair with a U only. If it has a G in it’s wobble posi8on, it can now bind to either C or U. That means that a codon of AAC OR AAU will bind to a tRNA that is GTT! So, one tRNA, carrying one specific amino acid (Asn ­ asparagine), can bind to two different codons! I’m not lying ­ look at your chart! Wobble pairing ­ more detailed explana8on! More reading this figure: if tRNA has U in the wobble posi8on, then it can pair with a A or G in the mRNA. That means that a codon of AAA OR AAG will bind to a tRNA that is UAA! So, one tRNA, carrying one specific amino acid (Lys ­lysine), can bind to two different codons! What about if the tRNA has an I in the wobble posi8on? That means a codon of AAC, AAA, or AAU should be coded by a tRNA carrying one specific amino acid (Asn ­asparagine). WHAT? Dr. S is lying to us ­ AAA isn’t Asn ­ it’s Lys! Your book is a liIle bit weird, here. A rarely appears in the codon wobble posi8on in real life. This is purely theore8cal (and confusing!). Ignore it ­ A will NOT be in the wobble posi8on! Wobble pairing ­ more detailed explana8on! Reading the boIom part of the figure: if mRNA has C in the wobble posi8on, then it can pair with a G or I in the tRNA. Basically, this is the reverse ­ the difference? Remember ­ you can’t have I in your mRNA sequence! That means that a codon of UUC will bind to a tRNA that has an an8codon sequence of either GAA or IAA! So, there are two tRNAs that could bind here. Both will be carrying Phe ­ phenylalanine. So, one codon can bind two different tRNA molecules. But, the end result doesn’t maIer ­ they will each have the same aa! What about if the mRNA has a U in the wobble posi8on? It can pair with A, G, or I in the tRNA. So, a codon of UUU will bind to a tRNA with an an8codon sequence of AAA, GAA, or IAA. There are three tRNAs that can bind here. All will carry Phe. Again, ignore A in the wobble posi8on! This is “never” seen in nature! Clicker ques8on! •  What are the wobble posi8ons? A.  B.  C.  D.  E.  3rd posi8on in mRNA codon 3rd posi8on in tRNA an8codon 1st posi8on in tRNA an8codon A & B A & C Clicker ques8on! •  What are the wobble posi8ons? A.  B.  C.  D.  E.  3rd posi8on in mRNA codon 3rd posi8on in tRNA an8codon 1st posi8on in tRNA an8codon A & B A & C More 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 Func8on rRNAs are essen8al components of ribosomes RNA structural complexity in the bacterial 16S rRNA molecule Structural role: acts as scaffold to define posi8on of the ribosomal protein. 3’ end contains the an8 ­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 inac8on areas (E, P, A), while most of the proteins are on the outside of the complex. The protein and RNA ­ 3D rota8onal view of the complex Transla8on: the process (eukaryo8c details) •  Ini8a8on: regulated assembly of the ribosome –  with ac8vated tRNAimet and mRNA –  Scanning to find start ­ •  Kozak consensus sequence (5’ ­ACCAUGG ­3’) •  Elonga8on: polypep8de chain elonga8on –  Entry of succeeding aminoacyl ­tRNAs –  Bond forma8on ­pep8dyltransferase reac8on –  Transloca8on of ribosome with respect to the mRNA •  Elonga8on factors ­ A site •  Pep8dyltransferase reac8on ­ P site •  GTP hydrolysis •  Termina8on: reading “STOP” –  Release and dissocia8on Transla8on ini8a8on: regula8on of ribosomal subunits eIF: eukaryo8c ini8a8on factors They help stabilize complexes! Aser transla8on, the 80S dissociates and the 60S and 40S subunits pair with eIFs to ini8ate another round of transla8on Step 1: Transla8on ini8a8on ­ preini8a8on complex forma8on Can only bind Met ­tRNAiMet when it is GTP bound ­ phosphoryla8on of eIF2 prevents GTP exchange Special ­ac8vated methionine aIached to tRNAiMet ONLY FOR INITIATION! Step 2: Transla8on ­ ini8a8on complex forma8on 5’ cap on mRNA is bound by eIF4 complex eIF4G associates with eIF3 Step 3: Transla8on ini8a8on eIF4B is architectural ­ it posi8ons the eIF4A helicase subunit so it can remove short regions of secondary structure in the RNA “scans” with energy from ATP hydrolysis Looking for start codons! When the complex finds AUG, GTP bound to eIF2 is hydrolyzed ­ irreversible! Stops scanning! Complex dissociates. Kozak sequence ­ ACCAUGG Step 4: Transla8on ini8a8on Small subunit unites with the the large 60S subunit ­ catalyzed by eIF5 and eIF6! This associa8on is accompanied by hydrolysis of a GTP bound to eIF5 ­ this is a proofreading step! Also makes it irreversible (so complex won’t fall off AUG is situated at P site while making large (named for polypep8de) proteins)! Transla8on: the process (eukaryo8c details) •  Ini8a8on: regulated assembly of the ribosome –  with ac8vated tRNAimet and mRNA –  Scanning to find start ­ •  Kozak consensus sequence (5’ ­ACCAUGG ­3’) •  Elonga8on: polypep8de chain elonga8on –  Entry of succeeding aminoacyl ­tRNAs –  Bond forma8on ­pep8dyltransferase reac8on –  Transloca8on of ribosome along mRNA •  Elonga8on factors ­ A site •  Pep8dyltransferase reac8on ­ P site •  GTP hydrolysis •  Termina8on: reading “STOP” –  Release and dissocia8on Transla8on: the animated version Also: check out hIp://www.youtube.com/watch?v=u9dhO0iCLww Step 1: Elonga8on Elonga8on factor (EF) 1a (bound to GTP) delivers aminoacyl ­ tRNAs to the A site (aminoacylated) Step 2: Elonga8on When the codon and an8codon match, GTP is hydrolyzed ­ this induces a conforma8onal change in the ribosome ­ the binding between aminoacyl ­tRNA is 8ght, and the GDP and Pi are released. Proofreading step! Conforma8onal change posi8ons the aminacylated ­tRNA close to the aminoacylated ­tRNA in the P site Step 3: Elonga8on Pep8dyltransferase reac8on between the two amino acids ­ catalyzed by the large rRNA, which orients the atoms correctly for the reac8on to occur Step 4: Elonga8on ­ ribosome transloca8on Ribosome moves one codon down the mRNA ­ once this has occurred, EF2 ­GTP is hydrolyzed. Moves to E site (exit) Proofreading! Irreversible ­ prevents the ribosome from going the wrong way or moving more than one codon at a 8me. Ribosomal polypep8de assembly The ribosome processively links aminoacylated ­tRNAs (ac8ve). Aser the amino acids are linked, the unacylated ­tRNAs exit through the E site. The polypep8de chain threads through a channel in the large ribosomal subunit. Note the different loca8on of mRNA compared to the polypep8de! Transla8on: the process (eukaryo8c details) •  Ini8a8on: regulated assembly of the ribosome –  with ac8vated tRNAimet and mRNA –  Scanning to find start ­ •  Kozak consensus sequence (5’ ­ACCAUGG ­3’) •  Elonga8on: polypep8de chain elonga8on –  Entry of succeeding aminoacyl ­tRNAs –  Bond forma8on ­pep8dyltransferase reac8on –  Transloca8on of ribosome along mRNA •  Elonga8on factors ­ A site •  Pep8dyltransferase reac8on ­ P site •  GTP hydrolysis •  Termina8on: reading “STOP” –  Release and dissocia8on Termina8on eRF (release factor) 1 directly recognizes and binds to the stop codon. Another proofreading step! eRF3 ­GTP works with eRF1 to cleave the pep8dyl ­ tRNA, which releases the protein chain. Powered by GTP hydrolysis! Chaperones will now help fold the polypep8de chain into a three dimensional configura8on to make a func8onal protein Termina8on NOTE: muta8ons in the mRNA sequence can inac8vate genes. One such example is by nonsense muta8ons (when a stop codon is introduced into the reading frame inappropriately). Example: codon for tyrosine (UAC) is mutated to a stop (UAG). This can some8mes be overcome by muta8ons in tRNA genes! How? The an8codon sequence of the mutant tRNATyr does not read GUA (stop), but reads CUA (tyrosine) instead! It can s8ll be coupled to the amino acid chain (only the an8codon is mutated). Not efficient, because the majority of proteins will s8ll be terminated. But, enough copies can be made to allow some protein to be made. If enough can be made, this tRNA muta8on has suppressed the mRNA muta8on. Transla8on: how can efficiency be increased? 1.  Transla8on of an mRNA molecule by mul8ple ribosomes 2.  Rapid recycling of ribosomal subunits aser disengagement from the 3’ end Poly A tail: nuclear export, transla8on, and stability of mRNA. The tail is shortened over 8me, and, when it is short enough, the mRNA is enzyma8cally degraded Polyribosomes ­ >1 ribosome transla8ng mRNA Circular mRNAs facilitate this process ­ helped by PABPI ­ this protein binds to eIF4G (think back to ini8a8on!) and polyA tail. Can we harness this knowledge to improve human health? •  Puromycin (an8bio8c) –  From Streptomyces alboniger –  Mimics 3’ end of an aminoacyl ­tRNA –  Enters the A site, and is incorporated into the growing polypep8de chain –  Because of it’s amide bonds (versus the normal ester linkage of the tRNA), it is more resistant to hydrolysis (which stalls the ribosome and causes dissocia8on and premature termina8on) –  Not selec8ve for eukaryotes or prokaryotes More drugs and poisons •  Tetracyclines –  Bind to 30S subunit, blocking A site and inhibi8ng binding of aminoacyl ­tRNAs –  Selec8ve for prokaryotes •  Streptomycin –  binds to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of Met ­tRNAiMet –  Selec8ve for prokaryotes •  Toxins –  Diptheria toxin •  Inac8vates eEF2 –  Ricin •  2 chains –  One chain depurinates the 28S rRNA loop, completely inac8va8ng the 60S subunit Clicker ques8on! You have performed a drug screen, searching for an agent that will selec8vely inhibit prokaryo8c protein synthesis. Which of the following quali8es do you not want this drug to have? A.  Inhibi8on of EF1α ­GTP hydrolysis B.  Direct binding to the A site of the ribosome C.  Binding and inhibi8on of the 40S subunit of the ribosome D.  Cause frameshis muta8ons in mRNA E.  Prevent EF2 ­GTP hydrolysis Clicker ques8on! You have performed a drug screen, searching for an agent that will selec8vely inhibit prokaryo8c protein synthesis. Which of the following quali8es do you not want this drug to have? A.  Inhibi8on of EF1α ­GTP hydrolysis B.  Direct binding to the A site of the ribosome C.  Binding and inhibi8on of the 40S subunit of the ribosome ­ the 40S ribosomal subunit is only found in eukaryotes ­ inhibi8ng this would cause ubiquitous problems in protein synthesis. D.  Cause frameshis muta8ons in mRNA E.  Prevent EF2 ­GTP hydrolysis Lecture summary •  Finishing transla8on –  tRNA structure and func8on –  Wobble basepairing –  rRNA and ribosomes –  The process: 3 steps •  ini8a8on –> elonga8on –> termina8on •  Inhibi8ng the process: toxins and pharmaceu8cals •  DNA replica8on –  Overview –  Ini8a8on –  Bi ­direc8onal synthesis and polarity –  Proofreading and repair DNA replica8on •  High fidelity copying of DNA •  Step ­wise 5’ ­3’ polymeriza8on of dNTP monomers •  Semi ­conserva8ve (versus conserva8ve) –  Both strands are templates –  Look over Messelson Stahl experiment on page 140 (and classic experiment .pdf)! •  Mul8ple enzymes par8cipate in this process Complica8ons of DNA replica8on •  Arise from two proper8es of DNA: –  The two complementary strands are an8parallel –  DNA polymerases can only add nucleo8des in a 5’ ­3’ direc8on •  DNA must be –  Unwound (helicases) –  Primed –  Ligated back together •  Specific enzymes perform these ac8vi8es DNA synthesis: dNTP polymeriza8on DNA replica8on •  Beginning the process –  The site: replica8on origin •  Sequences are A ­T rich •  Size and sequence is variable –  Helicases •  Unwind DNA at the origin –  Primase (a special RNA polymerase) •  Forms a short, complimentary RNA primer on both DNA strands •  Elonga8on by DNA polymerase –  Site of ac8on: replica8on fork Lecture summary •  Finishing transla8on –  tRNA structure and func8on –  Wobble basepairing –  rRNA and ribosomes –  The process: 3 steps •  ini8a8on –> elonga8on –> termina8on •  Inhibi8ng the process: toxins and pharmaceu8cals •  DNA replica8on –  Overview –  Ini8a8on –  Bi ­direc8onal synthesis and polarity – Proofreading and repair Next class! ...
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