1. The smooth minor groove face of adenosine makes it ideal for packing
The A-minor motif - 1
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
Nissen et al PNAS 98: 4899

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

3. The A-minor motif – adenosine preferred interactions
N3 and 2´OH
"outside
Nissen et al PNAS 98: 4899

4. The decoding problem
Ogle et al TIBS 28: 259

5. A two step recognition mode
Ogle & Ramakrishnan 2005 Ann. Rev. Biochem 74: 129

6. Phe-tRNAPhe (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. mant-GTP (fluorescent GTP derivative)
3´-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate
(structure for 2´/3´-O-(N-methylanthraniloyl)-[-thio] guanosine triphosphate,
triethyl ammonium salt)
Gromadski & Rodnina 2004 Mol. Cell 13: 191

8. 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. Example 1 Binding of EF-Tu-GTP-Phe-tRNAPhe (Prf16/17) to 70S ribosomes
poly(U) programmed
poly(A) programmed
Binding of
EF-Tu-GTP-Phe-tRNAPhe (Prf16/17)
to 70S ribosomes
Subsequent fitting using a
reasonable model for the reaction
yields kinetic parameters
Rodnina et al Biochemistry 33: 12267

10. Example 2 Binding of EF-Tu-mantGTP-Phe-tRNAPhe to 70S ribosomes
1: poly(U) programmed with Ac-Phe-tRNAPhe in P-site
2: poly(A) programmed with tRNALys in P-site
Rodnina et al EMBO J 14: 2613

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

12. Kinetics constants for inital selection
Gromadski & Rodnina 2004 Mol. Cell 13: 191

13. Initial selection vs. proofreading
Gromadski & Rodnina 2004 Mol. Cell 13: 191

14. Conformational changes of EF-Tu upon GTP hydrolysis
EF-Tu-GDP
EF-Tu-GTP
Molecular Biology of the Gene

15. The tRNA binding site is only present in EF-Tu-GTP
Molecular Biology of the Gene

16. Ovierview of structure
ASL: gold, U6 "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
3´AAG 5´ anticodon
Ogle et al Science 292: 899

17. 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. Interactions of A1493 at the first position
class I A-minor interaction, requires Watson-Crick base bair
Ogle et al Science 292: 899

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

20. Interactions of G530 at the third position
Mg2+
No Watson-Crick base pair required for interaction
Ogle et al Science 292: 899

21. Paromomycin induces similar changes
As streptomycin, paromomycin reduces
translational fidelity, but binding site is different
Ogle et al Science 292: 899

22. Near-cognate ASLs do not yield defined electron density
anticodon: 3´GAG 5´
no binding?
disordered binding?
Ogle et al Cell 111: 721

23. Electron density is obtained with paromomycin
A1492 and A1493 flipped out
G530 in anti conformation
Ogle et al Cell 111: 721

24. Conformation changes of 30S indicate binding of near-cognate ASL
Ogle et al Cell 111: 721

25. Conformation change are different in the presence of paromomycin
anticodon: 3´GAG 5´: first & third position wobble
Ogle et al Cell 111: 721

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

27. Cognate tRNA induces closing also without paromomycin
anticodon 3´AAG 5´: third position wobble
Ogle et al Cell 111: 721

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

29. 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. No well defined density for either syn or anti conformation of G530
Ogle et al Cell 111: 721

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

32. Cognate ASL always induces domain closure.
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. Energy penalties for wobble base pairs
Ogle et al Cell 111: 721

34. The hybrid model
Moazed & Noller 1989 Nature 342: 142

35. Cryo-EM of EF-Tu-tRNA complexes with the ribosome
kirromycin stalled
EF-Tu-GDP-tRNA
complex
side view
top view
mostly w/o EF-Tu
mostly with EF-Tu
Valle et al EMBO J 21: 3557

36. Kirromycin stalled complexes are slightly different
Valle et al EMBO J 21: 3557

37. Ternary complex crystal structure does not fit density of it at ribosome
Valle et al EMBO J 21: 3557

38. tRNA needs to be distorted
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
Valle et al EMBO J 21: 3557

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

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

41. 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. Phenotypes of CSD mutants
mutation maps in region of OTC gene
Gorini & Kataja 1964 PNAS 51: 487

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

44. restrictive and ram mutations
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.
Ogle & Ramakrishnan 2005 Ann. Rev. Biochem. 74: 129

45. restrictive and ram mutations may influence closure movement
Ogle & Ramakrishnan 2005 Ann. Rev. Biochem. 74: 129

46. The Hirsh suppressor (tRNATrp G24A) may increase flexibility
allows read-through
at UGA with Trp
Ogle & Ramakrishnan 2005 Ann. Rev. Biochem. 74: 129