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

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 it ideal for packinginto the minor groove side of RNA helices

N1, N3 and 2ÓH are available for hydrogen bonding


The A-minor motif – adenosine specific interactions


N3 and 2ÓH"inside"

N3 "inside"and 2ÓH"outside"

(For A-U noH-bond with N3)


The A-minor motif – adenosine preferred interactions


N3 and 2ÓH"outside


The decoding problem

Ogle et al. 2003 TIBS 28: 259


A two step recognition mode

Ogle & Ramakrishnan 2005 Ann. Rev. Biochem 74: 129


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


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)


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.


Example 1

poly(U) programmed

poly(A) programmed

Binding ofEF-Tu-GTP-Phe-tRNAPhe (Prf16/17)to 70S ribosomes

Subsequent fitting using a reasonable model for the reactionyields kinetic parameters

Rodnina et al. 1994 Biochemistry 33: 12267


Example 2

Binding ofEF-Tu-mantGTP-Phe-tRNAPhe

to 70S ribosomes

1: poly(U) programmed with Ac-Phe-tRNAPhe in P-site2: poly(A) programmed with tRNALys in P-site

Rodnina et al. 1995 EMBO J 14: 2613


Measurement of peptide bond formation


[3H]fMet-tRNA + ternary complex of [14C]Phe-tRNArapid quenching with 0.8 M KOH (similar to stopped flow)determination of dipeptide formed


Kinetics constants for inital selection



Initial selection vs. proofreading



Conformational changes of EF-Tu upon GTP hydrolysis

Molecular Biology of the Gene

EF-Tu-GDP EF-Tu-GTP


The tRNA binding site is only present in EF-Tu-GTP

Molecular Biology of the Gene


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

Ogle et al. 2001 Science 292: 899

3ÁAG 5´ anticodon


Changes upon binding of cognate tRNA

A1492 and A1493 flip out of internal loop of helix 44G530 goes from syn to anti conformation



Interactions of A1493 at the first position


class I A-minor interaction, requires Watson-Crick base bair


Interactions of A1492 and G530 at the second position


class II A-minor interaction of A1492interaction network requires Watson-Crick base pair


Interactions of G530 at the third position


Mg2+

No Watson-Crick base pair required for interaction


Paromomycin induces similar changes


As streptomycin, paromomycin reducestranslational fidelity, but binding site is different


Near-cognate ASLs do not yield defined electron density

anticodon: 3´GAG 5´

no binding?disordered binding?

Ogle et al. 2002 Cell 111: 721


Electron density is obtained with paromomycin

A1492 and A1493 flipped outG530 in anti conformation



Conformation changes of 30S indicate binding of near-cognate ASL



Conformation change are different in the presence of paromomycin


anticodon: 3´GAG 5´: first & third position wobble


Similar differences are observed for another near-cognate ASL


anticodon: 3ÁGG 5´: second & third position wobble


Cognate tRNA induces closing also without paromomycin


anticodon 3ÁAG 5´: third position wobble


Binding of first position wobble pair (with paromomycin)


lack of complementary surface (van der Waals interactions) dehydration of 2ÓHs required (no space for water) no compensation


Binding of second position wobble pair (with paromomycin)

gray/black indicates regular conformation of G-U wobble pair



No well defined density for either syn or anti conformation of G530



Binding affinities


Paromomycin increases the affinityof cognate ASL but not of near-cognate ASLs!

competition experiments with 70S ribosomes


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 costsfor fixing A1492/1493 are already payed by paromomycin binding.

Near-cognate ASL binding alone does not gain enough energyto pay cost for domain closure.

Only if part of the costs is payed by parmomycin, domain closureis possible.

The total gain of energy seems to be similar with andwithout paromomycin for near-cognate ASL binding.



Energy penalties for wobble base pairs



The hybrid model

Moazed & Noller 1989 Nature 342: 142


Cryo-EM of EF-Tu-tRNA complexes with the ribosome

Valle et al. 2002 EMBO J 21: 3557

mostly w/o EF-Tu mostly with EF-Tu

side view

top view

kirromycin stalledEF-Tu-GDP-tRNA

complex


Kirromycin stalled complexes are slightly different



Ternary complex crystal structure does not fit density of it at ribosome



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


Accomodation involves relaxation of tRNA


Faster for cognate tRNA due to restricted conformational space?


EF-Tu GTPase activation?


tRNA?sarcin-ricin-loop (SRL)?


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


Phenotypes of CSD mutants

Gorini & Kataja 1964 PNAS 51: 487

mutation maps in region of OTC gene


Streptomycin induces mistranslation in vitro

ribosomes + 20 amino acids, one labeled in each experiment

Davies et al. 1964 PNAS 51: 883

Ile: AUY, AUALeu: CUN, UURSer: UCN, AGY


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.


restrictive and ram mutations may influence closure movement



The Hirsh suppressor (tRNATrp G24A) may increase flexibility


Hirsh suppressorallows read-throughat UGA with Trp

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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