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Spezielle Genetik und Molekularbiologie SS 2006 The A-minor motif - 1 Nissen et al. 2001 PNAS 98: 4899 The smooth minor groove face of adenosine makes.

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Presentation on theme: "Spezielle Genetik und Molekularbiologie SS 2006 The A-minor motif - 1 Nissen et al. 2001 PNAS 98: 4899 The smooth minor groove face of adenosine makes."— Presentation transcript:

1 Spezielle Genetik und Molekularbiologie SS 2006 The A-minor motif - 1 Nissen et al PNAS 98: 4899 The smooth minor groove face of adenosine makes it ideal for packing into the minor groove side of RNA helices N1, N3 and 2´OH are available for hydrogen bonding

2 Spezielle Genetik und Molekularbiologie SS 2006 The A-minor motif – adenosine specific interactions Nissen et al PNAS 98: 4899 N3 and 2´OH "inside" N3 "inside" and 2´OH "outside" (For A-U no H-bond with N3)

3 Spezielle Genetik und Molekularbiologie SS 2006 The A-minor motif – adenosine preferred interactions Nissen et al PNAS 98: 4899 N3 and 2´OH "outside

4 Spezielle Genetik und Molekularbiologie SS 2006 The decoding problem Ogle et al TIBS 28: 259

5 Spezielle Genetik und Molekularbiologie SS 2006 A two step recognition mode Ogle & Ramakrishnan 2005 Ann. Rev. Biochem 74: 129

6 Spezielle Genetik und Molekularbiologie SS 2006 Phe-tRNA Phe (Prf 16/17) (fluorescent tRNA derivative) 1. Remove Y (wybutine) by acid treatment 2. React remaining aldehyde group at sugar with proflavine Gromadski & Rodnina 2004 Mol. Cell 13: 191

7 Spezielle Genetik und Molekularbiologie SS 2006 mant-GTP (fluorescent GTP derivative) Gromadski & Rodnina 2004 Mol. Cell 13: 191 3´-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate (structure for 2´/3´-O-(N-methylanthraniloyl)-[-thio] guanosine triphosphate, triethyl ammonium salt)

8 Spezielle Genetik und Molekularbiologie SS 2006 Stopped flow - Under pneumatic drive activation, the two small volumes of solutions are driven from high performance syringes through a high efficiency mixer. - The resultant mixture passes through a measurement flow cell and into a stopping syringe. - Just prior to stopping, a steady state flow is achieved. - As the solution fills the stopping syringe, the plunger hits a block, causing the flow to be stopped instantaneously. - Using appropriate techniques, the kinetics of the reaction can be measured in the cell.

9 Spezielle Genetik und Molekularbiologie SS 2006 Example 1 poly(U) programmed poly(A) programmed Binding of EF-Tu-GTP-Phe-tRNA Phe (Prf16/17) to 70S ribosomes Subsequent fitting using a reasonable model for the reaction yields kinetic parameters Rodnina et al Biochemistry 33: 12267

10 Spezielle Genetik und Molekularbiologie SS 2006 Example 2 Binding of EF-Tu-mantGTP-Phe-tRNA Phe to 70S ribosomes 1: poly(U) programmed with Ac-Phe-tRNA Phe in P-site 2: poly(A) programmed with tRNA Lys in P-site Rodnina et al EMBO J 14: 2613

11 Spezielle Genetik und Molekularbiologie SS 2006 Measurement of peptide bond formation Gromadski & Rodnina 2004 Mol. Cell 13: 191 [ 3 H]fMet-tRNA + ternary complex of [ 14 C]Phe-tRNA rapid quenching with 0.8 M KOH (similar to stopped flow) determination of dipeptide formed

12 Spezielle Genetik und Molekularbiologie SS 2006 Kinetics constants for inital selection Gromadski & Rodnina 2004 Mol. Cell 13: 191

13 Spezielle Genetik und Molekularbiologie SS 2006 Initial selection vs. proofreading Gromadski & Rodnina 2004 Mol. Cell 13: 191

14 Spezielle Genetik und Molekularbiologie SS 2006 Conformational changes of EF-Tu upon GTP hydrolysis Molecular Biology of the Gene EF-Tu-GDPEF-Tu-GTP

15 Spezielle Genetik und Molekularbiologie SS 2006 The tRNA binding site is only present in EF-Tu-GTP Molecular Biology of the Gene

16 Spezielle Genetik und Molekularbiologie SS 2006 Ovierview of structure ASL: gold, U 6 "mRNA": purple, helix 44: cyan, 530 loop: light green, helix 34: light blue, S12: tan, P-site tRNA mimic (helix 6 from neighbouring molecule): dark blue, P-site mRNA mimic (3´ end of 16S rRNA): dark blue Ogle et al Science 292: 899 3´AAG 5´ anticodon

17 Spezielle Genetik und Molekularbiologie SS 2006 Changes upon binding of cognate tRNA A1492 and A1493 flip out of internal loop of helix 44 G530 goes from syn to anti conformation Ogle et al Science 292: 899

18 Spezielle Genetik und Molekularbiologie SS 2006 Interactions of A1493 at the first position Ogle et al Science 292: 899 class I A-minor interaction, requires Watson-Crick base bair

19 Spezielle Genetik und Molekularbiologie SS 2006 Interactions of A1492 and G530 at the second position Ogle et al Science 292: 899 class II A-minor interaction of A1492 interaction network requires Watson-Crick base pair

20 Spezielle Genetik und Molekularbiologie SS 2006 Interactions of G530 at the third position Ogle et al Science 292: 899 Mg 2+ No Watson-Crick base pair required for interaction

21 Spezielle Genetik und Molekularbiologie SS 2006 Paromomycin induces similar changes Ogle et al Science 292: 899 As streptomycin, paromomycin reduces translational fidelity, but binding site is different

22 Spezielle Genetik und Molekularbiologie SS 2006 Near-cognate ASLs do not yield defined electron density anticodon: 3´GAG 5´ no binding? disordered binding? Ogle et al Cell 111: 721

23 Spezielle Genetik und Molekularbiologie SS 2006 Electron density is obtained with paromomycin A1492 and A1493 flipped out G530 in anti conformation Ogle et al Cell 111: 721

24 Spezielle Genetik und Molekularbiologie SS 2006 Conformation changes of 30S indicate binding of near-cognate ASL Ogle et al Cell 111: 721

25 Spezielle Genetik und Molekularbiologie SS 2006 Conformation change are different in the presence of paromomycin Ogle et al Cell 111: 721 anticodon: 3´GAG 5´: first & third position wobble

26 Spezielle Genetik und Molekularbiologie SS 2006 Similar differences are observed for another near-cognate ASL Ogle et al Cell 111: 721 anticodon: 3´AGG 5´: second & third position wobble

27 Spezielle Genetik und Molekularbiologie SS 2006 Cognate tRNA induces closing also without paromomycin Ogle et al Cell 111: 721 anticodon 3´AAG 5´: third position wobble

28 Spezielle Genetik und Molekularbiologie SS 2006 Binding of first position wobble pair (with paromomycin) Ogle et al Cell 111: 721 lack of complementary surface (van der Waals interactions) dehydration of 2´OHs required (no space for water) no compensation

29 Spezielle Genetik und Molekularbiologie SS 2006 Binding of second position wobble pair (with paromomycin) gray/black indicates regular conformation of G-U wobble pair Ogle et al Cell 111: 721

30 Spezielle Genetik und Molekularbiologie SS 2006 No well defined density for either syn or anti conformation of G530 Ogle et al Cell 111: 721

31 Spezielle Genetik und Molekularbiologie SS 2006 Binding affinities Ogle et al Cell 111: 721 Paromomycin increases the affinity of cognate ASL but not of near-cognate ASLs! competition experiments with 70S ribosomes

32 Spezielle Genetik und Molekularbiologie SS 2006 Reasons? Cognate ASL always induces domain closure. Sufficient energy gain to pay the energy costs for domain closure. Paromomycin should lower those costs, since e.g. the entropic costs for fixing A1492/1493 are already payed by paromomycin binding. Near-cognate ASL binding alone does not gain enough energy to pay cost for domain closure. Only if part of the costs is payed by parmomycin, domain closure is possible. The total gain of energy seems to be similar with and without paromomycin for near-cognate ASL binding. Ogle et al Cell 111: 721

33 Spezielle Genetik und Molekularbiologie SS 2006 Energy penalties for wobble base pairs Ogle et al Cell 111: 721

34 Spezielle Genetik und Molekularbiologie SS 2006 The hybrid model Moazed & Noller 1989 Nature 342: 142

35 Spezielle Genetik und Molekularbiologie SS 2006 Cryo-EM of EF-Tu-tRNA complexes with the ribosome Valle et al EMBO J 21: 3557 mostly w/o EF-Tumostly with EF-Tu side view top view kirromycin stalled EF-Tu-GDP-tRNA complex

36 Spezielle Genetik und Molekularbiologie SS 2006 Kirromycin stalled complexes are slightly different Valle et al EMBO J 21: 3557

37 Spezielle Genetik und Molekularbiologie SS 2006 Ternary complex crystal structure does not fit density of it at ribosome Valle et al EMBO J 21: 3557

38 Spezielle Genetik und Molekularbiologie SS 2006 tRNA needs to be distorted Valle et al EMBO J 21: 3557 fitting of tRNA from EF-Tu-GTP-tRNA complex fitting of A-site tRNA in 70S ribosome complex fitting of a chimera of both structures

39 Spezielle Genetik und Molekularbiologie SS 2006 Accomodation involves relaxation of tRNA Ogle & Ramakrishnan 2005 Ann. Rev. Biochem 74: 129 Faster for cognate tRNA due to restricted conformational space?

40 Spezielle Genetik und Molekularbiologie SS 2006 EF-Tu GTPase activation? Ogle & Ramakrishnan 2005 Ann. Rev. Biochem 74: 129 tRNA? sarcin-ricin-loop (SRL)?

41 Spezielle Genetik und Molekularbiologie SS 2006 Conditional streptomycin dependent (CSD) mutants CSD mutants grow well in rich medium CSD mutants require streptomycin for growth in minimal medium Gorini & Kataja 1964 PNAS 51: 487

42 Spezielle Genetik und Molekularbiologie SS 2006 Phenotypes of CSD mutants Gorini & Kataja 1964 PNAS 51: 487 mutation maps in region of OTC gene

43 Spezielle Genetik und Molekularbiologie SS 2006 Streptomycin induces mistranslation in vitro ribosomes + 20 amino acids, one labeled in each experiment Davies et al PNAS 51: 883 Ile: AUY, AUA Leu: CUN, UUR Ser: UCN, AGY

44 Spezielle Genetik und Molekularbiologie SS 2006 restrictive and ram mutations Ogle & Ramakrishnan 2005 Ann. Rev. Biochem. 74: 129 Restrictive mutations abolish read-through at leaky mutations. Map in rpsL gene (protein S12) required for streptomycin resistance. Restrictive mutation have a hyperaccurate phenotype in translation. ram mutations (ribosomal ambiguity) revert restrictive phenotypes. Second site mutations that change proteins S4 or S5. ram single mutants have increased read-through at nonsense mutations.

45 Spezielle Genetik und Molekularbiologie SS 2006 restrictive and ram mutations may influence closure movement Ogle & Ramakrishnan 2005 Ann. Rev. Biochem. 74: 129

46 Spezielle Genetik und Molekularbiologie SS 2006 The Hirsh suppressor (tRNA Trp G24A) may increase flexibility Ogle & Ramakrishnan 2005 Ann. Rev. Biochem. 74: 129 Hirsh suppressor allows read-through at UGA with Trp


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