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Exploring Biochemical Function of Macromolecules

by: Dr. Pablo Pollich

Exploring Biochemical Function of Macromolecules BIOCHEM 710

Marketplace > University of Wisconsin - Madison > Biochemistry > BIOCHEM 710 > Exploring Biochemical Function of Macromolecules
Dr. Pablo Pollich
GPA 3.75

David Brow

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David Brow
Class Notes
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This 9 page Class Notes was uploaded by Dr. Pablo Pollich on Thursday September 17, 2015. The Class Notes belongs to BIOCHEM 710 at University of Wisconsin - Madison taught by David Brow in Fall. Since its upload, it has received 71 views. For similar materials see /class/205191/biochem-710-university-of-wisconsin-madison in Biochemistry at University of Wisconsin - Madison.


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Date Created: 09/17/15
Biochem BMC 710 Biochemistry of catalytic RNAS Dave Brow amp Sam Butcher February 17 2009 lntrons are internal segments of RNA that are removed from a transcript during its maturation 5 3 Exon lntron Exon Exons are what is left over after introns are removed Exons may be proteincoding noncoding or a mix lntrons are found in premRNA pretRNA prerRNA presnRNA and presnoRNA Some snoRNAs come from premRNA introns There are 3 biochemical pathways for splicing 1 Protein endonuclease and ligase eukaryotic and archaeal tRNAs 2 Spliceosomal 2step transesteri cation nuclear premRNAs 3 Autocatalytic ribozymatic 2step trans esteri cation group I II and Ill introns Group II introns selfsplice by a twostep reaction identical to that catalyzed by the spliceosome Selfsplicing inlrons Spliceosamuaxalyud spiking dear of nu mrmA Groupl Group ll Splmeosome 1 3 2 gt4 H045 HM PM gt r xpt 1 Group II introns are found in bacteria and eukaryotic organelles Group II inrmn set indudes M ochondrlai Souspiking In vllm g 3 Twlnuons summm WI Fm mums m Lmupm mmqu Maw nipa mm W amalm mmnnns l awnmun sum Euglwlmd taimvmunn kkmd m Bmarial am cmmmm m W JGHMgollllwbacwllt 5mm J tanuhnclm m EmmiBriana hummus 1 Bonen amp Vogel 2001 Trends Genet 17322 y m m Sumo Secondary and tertiary structure elements of group II introns IIA EBS e Fedorova amp Zingler 2007 Biol Chem 388665 i o o r m w m 1 i ilhup39wwwhmmonLEduappiitaunnsmlnIdgirbmmarlnrmlgl v v Emmi Q 3 km Cayuu l UT Imam army msmn mr nmm Identifying secondary structure by phylogenetic base covariation Species 1 SYGUUCUGCGAAGAACSY Species 2 GUUCUGUGAAGAAC Species 31 Species 4 GAUCUGCGAAGAUC Species 51 Labeling RNA at 5 and 3 termini for biochemical experiments Phosphomlating the 5 end HO 5 o N T4poLyrr11leeotide 93410 5 O N w 5 y32PATP gt 393 ADP 0 0H 0 OH RNA RNA Li atin a labeled nucleotide C to the 3 end RNA 5 o N RNA 5 0 N m 532Ppcp T4 RNA ligase w AMP ppi OH OH ATP 393 OH bib o Cp pCp cytidine5 3 bisphosphate Identifying secondary structure by partial nuclease digestion 5 pGUUC 81 nuclease products pggggU 39 t d 39f39 sing e s ran speCI ic BGUUC pGUUC pGUUCUs V1 nuclease products pG doublestrand specificpGU GUU Chemical probes for RNA structure H H OO meo Ni 394 c 5 H30OSOCH3 Ca 5 quotquotc 596 crm dimethyl sulfate DMS Nw lt quot CJ 2 H0 CIL c quotfox c l T 0 v 39 0 0H Hi adenine uracil 39 A HM c lt H c C CBHs M quotdcNi 6 N c quotHr o diethyl pyrocarbonate DEPC guanine H cytosine Identifying secondary structure by partial chemical modification 5 GUUCUGCGAAGAAC3 DMS modification UUCUGCGAAGAAc GUUCU CGAAGAAC G GRk GUUCUGCGAAGAAC U A GUUCUGCGAAGAAC 8 GUUCUGCGAAgAAC U GUUCUGCGAAGAAC 35 pwwm kethoxal modification GUUCUC ECGAAGAAC GUUCUGC AAGAAC Mapping sites of chemical modification by reverse transcription modified RNA 5 3 anneal to a add Reverse Transcriptase Compementary 32p RT and dNTPs to make cDNA DNA ongonudeotide resolve products on a denaturing polyacrylamide gel DMS T U6A79GI 3 th6 g a 91quot l H electrophoresis C72 A73 A75 Note methylated G s don t stop RT unless backbone is cleaved by furthertreatments Identifying functional sites in RNA by modification and selection Eg ggg g 1 Lightly treat RNA solution to give random distribution of single hits gfegege 2 Incubate modified RNA with binding partner or in reaction buffer if ribozyme 3 Separate active and inactive RNA eg by gel electrophoresis or P 4 Map sites of modification in active and inactive populations Crosslinking 4thioU substituted RNA with near UV light s i HN o olt l 7 NH H N ON N gt300 nm UV H l H H SH 8 N H S a R H H N 4thiouracil vNPib NN Rib o 0 Rib s o Fab 0 N N HN N Nr i i gt H V N luvN N HZN f quot SH gt W R N N N 6thioguanine 7 R N O wwwm olgen m pg deagribolagbrim acom bedrcIrS4U html Group II ribozyme 3 structure Group II ribozyme 3 structure secondary structure Identification of 3 interactions W C Group II ribozyme 3 structure secondary structure Identification of 3 interactions nonWC Group II ribozyme 3 structure How are nonWC 3 interactions found secondary structure Identification of 3 interactions nonWC How are nonWC 3 interactions found Identification of nonWC 3 interactions were described in the following 3 landmark papers 1 Michel ampWesthof J Mol Biol 1990 2 Murphy amp Cech J Mol Biol 1994 3 Cate et al Science 1996 l Mall Biol I990 216 5357610 Modelling of the Threedimensional Architecture of Group I Catalytic Introns Based on Comparative Sequence Analysis Francois Michel and Eric Westhof2 Used sequence alignments of 87 Group catalytic introns Found covariation between GAAA tetraloops and tandem CG pairs among others Constructed an allatom model of intron core Table 8 Covariation between the L9 terminalloop and P5 bp 2 and 3 P9 Canonical L9 terminal loop 2 1 Hal Binl W94 236 49763 GAAA Tetraloop and Conserved Bulge Stabilize Tertiary Structure of a Group I Intron Domain Felicia L Murphy and Thomas R CechT Probed structure of group I catalytic intron w FeEDTA Found point mutation in P5b GAAAtetraloop disrupts 3 interaction with another helix P6a dentified a stable subdomain P46 Subgroups GNAA GYGA P5 bp 2 and 3 bp 3 UA bp 2 CG 7 7 CG CG 18 0 Other CG 9 2 70 Other I 0 Other Other 0 2 2 JS SA PulP JLAA GAGA MAG l L39CG Pnirul AIKEl39 uu unso n3 ousa u 350 121 um r3200 mas m7 1in Gm m m mmn 59 15mm SUE713520 September 1996 Prev Table 0 Contents Next gt VDi273nD5282pp1678 1685 DOI 101 ZSscience 27352811578 RBEARCH ARTICLES Crystal Structure of a Group I Ribozyme Domain Principles of RNA Packing Jamie H Cate Anne R ooding Elaine Podell Kaihong Zhou Barbara L Golden Craig E Kundrol Thomas R Ceth I Jennifer A Doudna A Comparison of model vs crystal structure Group II ribozyme 3 structure How are nonWC 3 interactions found secondary structure Identification of 3 interactions nonWC Group II ribozyme 3 structure secondary structure model ofthe catalytic core Swisher Duane Su amp Pyle EMBO 2001 20 2001 Group II intron D5 and D6 structure Comparison of U6 ISL and Group II Domain 5 A NMR structures C A U G A U A 8 8 c AU 8 U U A 62X G C GC B 830 A39 C CG B72 880 884 815 820 0 AU GU 539C A A l i l 1 Let s J A Lg GU u ccu LJCGG r n GAG39 l GUA u Cleo 39 u CA A GGG GGCCV ll CUUGGCAUJ 5 3 5 3 I A A H JCV A 863 860 852 348 U6 ISL Domain 5 D Wm Zhang and Doudna 2002 Science 295556220488 Table 1Xray data collection MAD phasing and structure re nem t Space group P61 uni cell a 0 c 3 89 A native a b 9168 A c 24165 A bromo 39 39 833 an he cosine ofthe error in phase angle for a speci c re ection Re nemen o quot 39 ions Numbers after slash refer to the highest resolution bin Parameter Native Bromo at wavelength lambda 1 lambda 2 lambda 3 lambda 4 Crystallographic data Wavelength A 11 09200 194 09273 09020 Resolution A 3030 3035 3 3 3035 Not a 3 A mpleteness 898 835475 Rsym 541430 4923 44303 37324 4034o structure lldelta 23 0 22 52 7 2282 5 25 72 3 2312 0 Redundanc 155 287 287 MAD phase determination lambda 1 lambda 2 lambda 4 Resolution A 3036 3036 3036 Rcullisdagger 07573 07225 07754 P asing powerddagger 08398 13290 10340 Re nement Resolution A 3030 st 243 Rfree 2 rmsd bonds A 0006591 Average Bfactor A2 99 55 gle Ouch In LI T Group II introns can catalyze 5 ss hydrolysis as well as branching Ribozyme kinetics can be studied like enzyme kinetics 1 Main palhwly h Alimum pathway 5 Enquot HOH s39 d 2 04 20 e101 23 p A E E sspumsiuj mo D123 I352 3 5 a t Transeslan calion I Hydrolysis 3 V r E 39 Ds v39 05 ou A A Yranss5levlhcauun eXD123 D5 T exD123D5 fexD123D5 d chem F DH Salaam 92 2 3 4 5 7 3 9 2 3 exD123 372 quot l lt D123 06Iusslcaldomalnvl 4mm organelles 650 605 7 D123 4 mlch w quotas u c A quot 350 quotquotV cAciu g gu g uSS c g m can 35 e Nuter HIquotIRMA Ag quotam1 Aucuucwc dr susa am 4 ex u cu 23v 27 39 nadlZ EWCWGGA UAUG mc Note that in this case D5 is the catalyst ribozyme and is in excess mm m Determination of kchem requires saturating D5 ex D123 w 20 so u so nmemln knb mm 0 mm anon anon was soon sum DWnMi


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