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Alan Weiner BIOCHEM 530 Monday, EEB 037 October 6, 2014 RNA structure and function.

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Presentation on theme: "Alan Weiner BIOCHEM 530 Monday, EEB 037 October 6, 2014 RNA structure and function."— Presentation transcript:

1 Alan Weiner BIOCHEM 530 Monday, EEB 037 October 6, 2014 RNA structure and function

2

3 Crick's "Central Dogma"? DNA makes RNA makes protein

4 Crick's "Central Dogma"? DNA makes RNA makes protein or

5 Crick's "Central Dogma"? DNA makes RNA makes protein ( us) ribosome spliceosome

6 RNA is more than a structure or informational sequence… key steps in the "Central Dogma" are catalyzed and regulated by RNAs DNA mRNA protein mRNA splicing catalyzed by small nuclear RNAs (snRNAs) translation of mRNAs catalyzed by ribosomal RNAs (rRNAs) and tRNAs mRNA transcription regulated by long noncoding RNAs (lncRNAs) translation regulated by microRNAs (miRNAs) chromatin structure regulated by small interfering RNAs (siRNAs and piRNAs)

7 RNA is more than a structure or informational sequence… key steps in the "Central Dogma" are catalyzed and regulated by RNAs DNA mRNA protein mRNA splicing catalyzed by small nuclear RNAs (snRNAs) translation of mRNAs catalyzed by ribosomal RNAs (rRNAs) and tRNAs mRNA transcription regulated by long noncoding RNAs (lncRNAs) translation regulated by microRNAs (miRNAs) chromatin structure regulated by small interfering RNAs (siRNAs and piRNAs) defensive RNAsbiosynthetic and regulatory RNAs

8 The principles of RNA folding

9 tertiary interactions very important distances misleading continuous coaxial stack tRNA is built from two structurally independent domains tethered together

10 A space-filling model of a folded tRNA looks a lot like a globular protein

11 RNA folding (music and colors added) http://www.youtube.com/watch?v=PWETG3B6wek; Penchovsky and Breaker (2005) Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes. Nat Biotechnol 23, 1424 experimentally determined (2D) structure

12 tRNA (76 nt) Group I ribozyme P4-P6 domain (160 nt) 70S ribosome (4530 nt total plus 50 proteins), 3 rRNAs, 2 tRNAs, 1 mRNA 1974 to 2005: tRNA to Group I introns to the ribosome HN Noller (2005) RNA Structure: Reading the Ribosome. Science 309, 1508-1514.

13 1974 to 2005: tRNA to Group I introns to the ribosome tRNA (76 nt) Group I ribozyme P4-P6 domain (160 nt) 5S RNA tRNAs mRNA LSU proteins 23S rRNA 16S rRNA SSU proteins docking kissing, tethering, coaxial stacking 70S ribosome (4530 nt total plus 50 proteins), 3 rRNAs, 2 tRNAs, 1 mRNA

14 Coaxial stacking of helices in 16S rRNA and tRNA and the RNA folding problem! HN Noller (2005) RNA Structure: Reading the Ribosome. Science 309, 1508-1514. TCTC DHU AC CCA

15 Tetraloops are magical, unexpected, evolutionarily conserved, tight packings of loops containing only four nucleotides Baumruk et al. (2001) Comparison between UUCG and CUUG tetraloops: thermodynamic stability and structural features analyzed by UV absorption and vibrational spectroscopy. Nucleic Acids Res 29, 4089–4096 2’ endo syn A-form helix 2’ endo

16 Hoogsteen base pairs use the top edge of the purine base Watson-Crick base pairs Hoogsteen base pairs Ghosal and Muniyappa (2006) BBRC 343, 1-7

17 Aishima et al. (2002) Nucleic Acids Res 30, 5244–5252. Yeast MATα2 homeodomain bound to DNA: a Hoogsteen base pair embedded in (almost) undistorted B-DNA A

18 HN Noller (2005) RNA Structure: Reading the Ribosome. Science 309, 1508-1514. 1974 to 2005: tRNA to Group I introns to the ribosome tRNA (76 nt) Group I ribozyme P4-P6 domain (160 nt) 5S RNA tRNAs mRNA LSU proteins 23S rRNA 16S rRNA SSU proteins docking 70S ribosome (4530 nt total plus 50 proteins), 3 rRNAs, 2 tRNAs, 1 mRNA kissing, tethering, coaxial stacking

19 "A-minor" interactions: a lock-and-key between (usually) an adenosine and the minor groove of a Watson-Crick pair Type II Type I HN Noller (2005) RNA Structure: Reading the Ribosome. Science 309, 1508-1514.

20 HN Noller (2005) RNA Structure: Reading the Ribosome. Science 309, 1508-1514. black, real; red, very likely real; (conformation good, almost docked); blue, intersubunit bridge contacts A-minor interactions in 16S rRNA as seen in the 3.0 Å crystal structure of the Thermus thermophilus 30S ribosomal subunit

21 HN Noller (2005) Science 309, 1508-1514. How the ribosome packs two helices in 16S rRNA at right angles to each other

22 RNA 3D Structure Analysis and Prediction, Nucleic Acids and Mol Biol 27, 2012 (Leontis and Westhof, eds)

23 HN Noller (2005) RNA Structure: Reading the Ribosome. Science 309, 1508-1514. 1974 to 2005: tRNA to Group I introns to the ribosome tRNA (76 nt) Group I ribozyme P4-P6 domain (160 nt) 5S RNA tRNA mRNA LSU proteins 23S rRNA 16S rRNA SSU proteins docking 70S ribosome (4530 nt total plus 50 proteins), 3 rRNAs, 2 tRNAs, 1 mRNA kissing, tethering, coaxial stacking

24 All essential parts of the translation apparatus are made of RNA mRNA 30S mRNA binding 50S peptidyltransferase center EAP 5'3' and translation requires large movements of large molecules in close quarters aa 1 aa 2 aa 3 Ramakrishnan et al. (2006) Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935-1942 tRNA

25 RNPs (ribonucleoprotein complexes) you may know… ribosome (SSU and LSU) signal recognition particle (SRP) small nuclear ribonucleoprotein particles (snRNPs) small nucleolar ribonucleoprotein particles (snoRNPs) Francis Crick asked a dangerous question using the forbidden W word (why?) instead of the publicly acceptable H word (how?) but long ago, in 1968…

26 30S = 21 proteins + 1542 base ribosomal RNA 50S = 34 proteins + 2904 base ribosomal RNA Crick argued in 1968 (J Mol Biol 38, 367-379): The ribosome is a giant ribozyme! 1. Why is ribosome made half of protein and half of RNA? 2. What is job of ribosome? To make protein. 3. Therefore, the first ribosome had NO proteins to help out. 4. Therefore, the RNA itself must have been catalytic. 5. If RNA can be catalytic, RNA could have copied itself, functioning both as genome AND as replicase. 6. Therefore, the first "living molecule" could have been RNA! Gilbert carried this idea further in 1986 (Nature 319, 618) by coining a magical phrase, "The RNA World."

27 Lieberman et al. (2000) J Mol Biol 297, 1129 The large (23S) and small (16S) ribosomal RNAs have highly conserved secondary and tertiary structures the peptidyltransferase center on 23S rRNA is especially highly conserved; it is the locus of many antibiotic resistance mutations; and it is preferentially inactivated by RNA- modifying reagents

28 orange = sugar phosphate backbone white = nucleotide bases blue = proteins red = substrate analog Large (50S) ribosome subunit from Haloarcula marismortui, an archaeal halophile from the Dead Sea TA Steitz and PB Moore (2003) TIBS 28, 411–418 L6 space-filling model L11 protein backbone L1

29 orange = sugar phosphate backbone white = nucleotide bases blue = proteins red = substrate analog TA Steitz and PB Moore (2003) TiBS 28, 411-418 L6 space-filling model L11 protein backbone Large (50S) ribosome subunit from Haloarcula marismortui, an archaeal halophile from the Dead Sea L1

30 orange = sugar phosphate backbone white = nucleotide bases blue = proteins red = substrate analog TA Steitz and PB Moore (2003) TiBS 28, 411-418 L6 space-filling model L11 protein backbone Large (50S) ribosome subunit from Haloarcula marismortui, an archaeal halophile from the Dead Sea L1

31 The ribosome is a ribozyme… there are no proteins within 18 Angstroms of the active site A space-filling model of the peptidyltransferase center, with substrate analog (puromycin) in blue, RNA in red, and proteins in gray Steitz and Moore (2003) TIBS 28, 411-418; for update, see Beringer and Rodnina (2007) Mol Cell 26, 311-321

32 Substrate-assisted peptidyl transfer The  -amino group of the incoming aminoacyl-tRNA attacks the ester carbon of the resident peptidyl-tRNA to yield a tetrahedral intermediate (A). The intermediate breaks down to deacylated tRNA and elongated peptidyl-tRNA through a proton shuttle mechanism involving either the 2' hydroxyl of A76 on the peptidyl-tRNA substrate (B and C) or water molecules that interact with the 2' and 3' hydroxyls of A76 (D and E). Note that converting the 2' hydroxyl of A76 to a 2' deoxy reduces the rate of peptidyl transfer by 10 6 consistent with this proton shuttle mechanism. Schmeing, Strobel, Steitz et al. (2005) Mol Cell 20, 437-448 2'

33 Many antibiotics target overlapping regions of the peptidyltransferase center, or block the exit tunnel for the growing polypeptide. TA Steitz and PB Moore (2003) TIBS 28, 411-418

34 Many antibiotics interact with the large ribosomal RNA that surrounds the peptidyltransferase center backside view frontside view

35 A molecular fossil is any modern molecule whose structure or function provides a clue to its evolutionary history. Molecular fossils should not be confused with fossil molecules (DNA or insects preserved in amber) or "living fossils" (a slowly evolving organism like the deep-sea lobe-finned Coelacanth). Molecular fossils Weiner and Maizels (1987) PNAS 84, 7383-7387

36 If RNA can be both catalyst and genome, RNA could replicate itself… the first "living" molecule! catalytic (+) strand template (–) strand catalytic (+) strand copies unfolded template (–) strand two catalytic (+) strands and original template (–) strand Lawrence and Bartel (2005) New ligase-derived RNA polymerase ribozymes. RNA 11, 1173-1180; Shechner and Bartel (2011) The structural basis of RNA-catalyzed RNA polymerization. Nat Struct Mol Biol 18, 1036-1042; Shechner et al. (2009) Crystal structure of the catalytic core of an RNA- polymerase ribozyme. Science 326, 1271-1275; Bagby et al. (2009) A class I ligase ribozyme with reduced Mg 2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions. RNA 15, 2129-2146. precursors

37 Nucleic acids are plausible prebiotic condensation products

38 Nucleic acids are plausible prebiotic condensation products generated by repeated dehydrations uracil O OH P O O = O = 5' – H 2 O, glycosidic bond – H 2 O, phosphoester bond (ribose,  phosphate) ribose PP   – H 2 O, phosphoanhydride bonds ( ,  and ,  ) HN N

39 Whoops, right impulse, wrong concept! Szostak (2009) Origins of life: Systems chemistry on early Earth. Nature 459, 171-172 [News and Views] What Nature probably did… What modern salvage pathways and "prebiotic" chemists do…

40 "Systems chemistry on early earth"… proposed by Oparin in 1938, tested by Miller in 1953 as his PhD Thesis!

41 "Systems chemistry on early earth"… Options for pyrimidine ribonucleotide assembly Powner et al. (2009) Nature 459, 239-242 small molecules cyclic intermediates hydrolysis or phosphorolysis products old-fashioned chemical intuition new-fangled systems pathway

42 to be continued....

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