RNA Part I: Chemical Structure of RNA

Transcription

1 RA Part I: Chemical Structure of RA

2 Structural differences between RA and DA

3 Resistance of phosphate esters to basic hydrolysis

4 The 2 - group of RA facilitates chemical cleavage in aqueous a by forming a cyclic phosphate ester

5 Ribonucleases rapidly degrade RA: S2-like attack on phosphate

6 C to U mutation: the thymine methyl group allows the repair machinery To distinguish dt da from du dg Pairs with G Pairs with A!

7 RA adopts A-form helices primarily because of the C.3 endo pucker of the ribose sugar. A form B-form Z-form The major groove of the A-form helix is deep and narrow, the minor groove is wide and shallow.

8 Common RA Secondary Structures

9 RA folds into complex globular structures DA ammerhead ribozyme tra

10 Formation of hairpin (stem-loop) structures by intramolecular base pairing UCG typically forms the loop Structure.

11 Viral RA tertiary structure

12 Primary, Secondary, and Tertiary structure of tra

13 RA Part 2: Chemical Synthesis of RA

14 Monomeric units with 2 protecting group MMTr B R=Si(t-Bu)Me 2 (TBS) P R R=C 2 Si(iPr) 3 (TM) C Silyl ethers are easily cleaved with fluoride ion (TBAF) TBS ethers are sterically bulky and slow the rate of the coupling reaction, decreasing yields in some cases TBS ethers are also sensitive to the basic conditions needed in base deprotection TM ethers overcome these limitations

15 Synthesis of Monomers with Acylated bases

16 Standard deprotection/coupling/capping/oxidation cycle

17 Two step deprotection: basic conditions/fluoride

18 RA polymerases create new strands of RA

19 Inhibitor of RA polymerase

20 Transcription of mra involves many proteins and many regions of DA (Sequences rich in A and T)

21 Major Groove Recognition of DA by Proteins minor groove major groove GC4 Zif268 Lambda repressor Sequence Specific Contacts Arg R C 2 Asp R Asn, Gln R Thr, Ala, (Lys, Tyr) C 2 C 2 R major groove C C C C C 3 C 3 hydrophobic interaction R G C R R A T R

22 TBP: a minor groove binding transcription factor More weakly stacked A T base pairs makes TATA box more flexible

23 Transcription factors bind to DA with high sequence specificity Lac repressor DA sequence mismatch Lac repressor DA sequence match Upon identifying the match sequence, the hinge region forms An α-helix and bends the DA

24 Bacterial gene expression

25 Transcription factors Activators Repressors RAPs GTP cap, methylation (spliceosomes + ribozymes) Poly-A binding protein guides to riboso Ribosome rra, tra uman gene expression

26 The 5 -ends of mra are capped and methylated with SAM

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28 RA Part 3: Small molecule Binding of RA

29 Early Examples of Small Molecule-RA Binding guanosine R G264 C311 R 2 C arginine R G264 2-aminopurine C311 R R A254 U311 R 2 citrulline R A264 C U311 R Amidine and guanidine functional groups useful in the recognition f A and G!

30 Aminoglycosides are naturally occuring aminosugars that Bind to RA primarily by electrostatic interactions. They Associate with A-form helices in regions where there is a distortion (single base pair bulge) 2 R= eomycin B R 2 R= eomycin-thymine conjugate 2 aminoglycoside binding site A C U C A G U A U G C U A G C U A G C C G G U G C tau exon 10 splicing regulatory element binding site for Mitoxantrone Conjugate designed to target the bulged A base

31 eomycin nestled in the major groove of RA near a 2 bp bulge site

32 Tobramycin in the major groove of RA near a 2bp bulge

33 Intercalators planar aromatic moeities capable of inserting in the base-pair stack of RA again occuring at regions in which the helix is distorted by an internal loop or bulge glycoside g site A C U C A G U A U G C U A G C U A G C C G G U G C tau exon 10 splicing regulatory element binding site for Mitoxantrone Cationic side chains in Major groove Mitoxantrone Ethidium bromide, DAPI, aminoquinolines, netropsin are also bulge binders

34 bulged A Major Groove Minor Groove

35 The eomycin-acridine conjugate has high affinity for A-form nucleic acids: RA, GC-rich DA 2 eo-ac binding site eomycin-acridine conjugate S A UG G G CGCAGCGU C AC A G G GCGUCGCA U single uridine base bulge RRE G A A U 2 Conjugate binds distorted helical region by intercalation and major groove binding

36 elix-threading peptides place groups in the major and minor grooves of RA simultaneously weaker intercalator 2 2 helix threading peptides K D =20 nm U C U A C A U G C G C C G G C C G A A G C U U G C G C U A G U A U C G binding site for helix-threading peptides K D =208 nm Single base pair loop required for binding; no binding observed when loop was removed.

37 Janus-face ligands recognize U-U (RA, or T-T, DA) mismatches CUG repeats implied in myotonic dystrophy

38 Deoxystreptamine dimers selectively bind RA hairpin loops Kd µm µm µm µm

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41 The massive ribosome consists of proteins and RA: rra and tra

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43 Attachment of amino acid to 3 -adenine tra structure

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45 Amide bond formation promoted by a ribosomal adenine base

46 pseudouridine

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