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When there are more than one secondary structure exist Pseudoknot and kissing hairpins A pseudoknot structure is conserved in telomerase RNA component Some of the human disease related telomerase RNA mutants also locate within the PK region The PK of Human Telomerase RNA component L2 L1 Base triples between loops and stems in helical junction region The triple-helix region of yeast telomerase RNA is in close proximity to the 3’ end of the DNA substrate The RNA component of human telomerase can be reconstituted in a 2/3-pieces approach The 2’OH group in the triple-helix structure of human telomerase RNA is important for catalysis The 2’OH group of A176 in the triple-helix structure of human telomerase RNA is important for catalysis Ribosomal -1 frameshift Programmed -1FS requires two in cis RNA signals with a spacer of 5-7 nts between them Slippery site Stimulator Translation with -1 frameshift 3’ U C C G A G C GAAA AGGCUCG G U G G C A U C G G A A U U A -1 shift G C G C G C 5’ UAU UUA AAC GGG UUU UUGC -1 frame-shifting occur for the translation of SARS corona virus RdRp (n.t 13354-16091) 3CL-Pro cleavage A slippery sequence site stop CAG TCT GCG GAT GCA TCA ACG TTT TTA AAC GGG TTT GCG GTG TAA GTG CAG CCC GTC TTA CAC CGT Q S A D A S T F L N GCG GCA CAG GCA CTA GTA CTG ATG TCG TCT ACA GGG CTT TTG ATA TTT ACA ACG AAA AAG TTG CTG GTT TTG CAA AGT TCC TAA AAA CTA ATT GCT GTC GCT TCC AGG AGA AGG ATG AGG AAG GCA ATT TAT TAG ACT CTT ACT TTG TAG TTA AGA GGC ATA CTA TGT CTA ACT ACC AAC ATG AAG AGA CTA TTT ATA ACT TGG TTA AAG ATT GTC CAG CGG TTG CTG TCC ATG ACT TTT TCA AGT TTA GAG TAG ATG GTG ACA TGG TAC CAC ATA TAT CAC GTC AGC GTC TAA CTA AAT ACA CAA TGG CTG ATT TAG TCT ATG CTC TAC GTC ATT TTG ATG AGG GTA ATT GTG ATA CAT TAA AAG AAA TAC TCG TCA CAT ACA ATT GCT GTG ATG ATG ATT ATT TCA ATA AGA AGG ATT GGT ATG ACT TCG TAG AGA ATC CTG ACA TCT TAC GCG TAT ATG CTA ACT TAG GTG AGC GTG TAC GCC AAT CAT TAT TAA AGA CTG TAC AAT TCT GCG ATG CTA TGC GTG ATG CAG GCA TTG TAG GCG TAC TGA CAT TAG ATA ATC AGG ATC TTA ATG GGA ACT GGT ACG ATT TCG plus 1 slippery site TCT GCG GAT GCA TCA ACG TTT TTA AAC G GGT TTG CGG TGT AAG TGC AGC CCG TCT TAC ACC GTG Q S A D A S T F L Stop in +1 frame CGG CAC AGG CAC TAG TAC TGA TGT CGT CTA CAG GGC TTT TGA TAT TTA CAA CGA AAA AGT TGC TGG TTT TGC AAA GTT CCT AAA AAC TAA TTG CTG TCG CTT CCA GGA GAA GGA TGA GGA AGG CAA TTT ATT AGA CTC TTA CTT TGT AGT TAA GAG GCA TAC TAT GTC TAA CTA CCA ACA TGA AGA GAC TAT TTA TAA CTT GGT TAA AGA TTG TCC AGC GGT TGC TGT CCA TGA CTT TTT CAA GTT TAG AGT AGA TGG TGA CAT minus 1 slippery site CAG TCT GCG GAT GCA TCA ACG TTT TTA AACGG GTT TGC GGT GTA AGT GCA GCC CGT CTT ACA CCG Q S A D A S T F L N R V C G V S A A R L T P TGC GGC ACA GGC ACT AGT ACT GAT GTC GTC TAC AGG GCT TTT GAT ATT TAC AAC GAA AAA GTT GCT GGT TTT GCA AAG TTC CTA AAA ACT AAT TGC TGT CGC TTC CAG GAG AAG GAT GAG GAA GGC AAT TTA TTA GAC TCT TAC TTT GTA GTT AAG AGG CAT ACT ATG TCT AAC TAC CAA CAT GAA GAG ACT ATT TAT AAC TTG GTT AAA GAT TGT CCA GCG GTT GCT GTC CAT GAC TTT TTC AAG TTT AGA GTA GAT GGT GAC ATG GTA CCA CAT ATA TCA CGT CAG CGT CTA ACT AAA TAC ACA ATG GCT GAT TTA GTC TAT GCT CTA CGT CAT TTT GAT GAG GGT AAT TGT GAT ACA TTA AAA GAA ATA CTC GTC ACA TAC AAT TGC TGT GAT GAT GAT TAT TTC AAT AAG AAG GAT TGG TAT GAC TTC GTA GAG AAT CCT GAC ATC TTA CGC GTA TAT GCT AAC TTA GGT GAG CGT GTA CGC CAA TCA TTA TTA AAG ACT GTA CAA TTC TGC GAT GCT ATG CGT GAT GCA GGC ATT GTA GGC GTA CTG ACA TTA GAT AAT CAG GAT CTT AAT GGG AAC TGG TAC GAT TTC 1 S1 S2 S1C 50 Avian TTTAAACGGG TACG GGGTAG CAG T...GAG GCTCGG CTGA TACCC CTTGC BOVINE TTTAAACGGG TTCG GGGTAC GAG TGTAGAT GCCCGT CTCG TACCC TGTGC SARS TTTAAACGGG TTTG CGGTGT AAG T...GCA GCCCGT CTTA CACCG TGCGG HUMAN TTTAAACGAG TCCG GGGCTC TAG TGCCGCT CGACTA GAGC CCTGT AATGG CONSENSUS TTTAAACGGG TTCG GGGTAC –AG TG--G-T GCCCGT CTGA –ACCC TGTG- 51 S2C 100 Avian TAGTGGATGT GATCCTGATG TTGTAAAGCG AGCCTTTGAT GTTTGTAATAAGG BOVINE CAGTGGTTTA TCTACTGATG TACAATTAAG GGCATTTGAT ATTTGCAATG~~~ SARS CACAGGC ACT AGTACTGATG TCGTCTACAG GGCTTTTGAT ATTTACAACGAAA HUMAN TACAGACATA GATTACTGTG TCCGTGCATT TGACGTTTAC AATAAAGATG~~~ CONSENSUS -A--GGC-T- GATACTGATG TC-TATAAAG GGCCTTTGAT ATTT-CAATGA- - Sequence co-variation for base-pairing between two potential duplex region S1 and S2. An atypical PK with a third stem region formed within loop 2. L2 Probing of secondary structure by ribonuclease mapping: RNase T1- prefer unpaired G RNase A- prefer unpaired C/U RNase T2-prefered single-stranded RNase V- prefer duplex region All three stem regions contribute to the -1FS activity. The secondary structures of mRNA need to be unwound into single-stranded codon information for ribosome decoding. S3, S4, S5 As Helicase ? -1FS efficiency vary among different pseudoknots A different kind of RNA duplex Story of RNA kissing-hairpin and retroviral RNA dimerization Kinetic can play important role in regulation of cellular process when a time window is set for a specific event ROM Structure-specific RNA-binding A structure-specific RNA-binding protein B-form DNA A-form RNA A compressed and blocked major groove. A distroted minor groove surface with realigned 2’OH Similar RNA-RNA interactions also occur in virus The dimerization initiation site of HIV and MMLV retroviral RNA genome Positive-stranded dimeric RNA genome in a 5’- parallel-parallel orientation. Kissing-loop Extended duplex The NC protein can facilitate complete dimerization of kissing complex DIS of Moloney Murine Leukemia Virus Complementary How can stem-loops without self complementary sequence can facilitate intermolecular recognition? Cross-strand stacking Monomer-Dimer transition Zinc-finger mediated NC-viral RNA interactions NC binds to dimeric forms of DIS-1 and DIS-2 Dimerization-induced exposure of NC protein binding-site HCV Loop-Loop interactions in plant viruses RNA acting as Enzyme HDV ribozyme Cofactor-dependent glmS ribozyme Role of 2’OH in hydrolysis of RNA Metal ion catalysis vs general acid-base catalysis Concerted general acid-base catalyzed RNA cleavage reaction by RNase A. The pKa of Imidazole ring of Histidine are close to neutral in physiological pH Small Ribozymes Hammerhead Hairpin HDV Leadzyme Acid-base catalysis in HDV ribozyme Nature 395, 567-574 (1998) Science 286,123-126 (1999) The genomic and antigenomic strand HDV ribozymes can fold into similar secondary structure. A pre-organized rigid fold An intricate fold that buries the active site deep within a catalytic cleft Two parallel helical stack A solvent inaccessible cleft surrounded by packed helices The interactions that produce P1.1 constrain the ribozyme into its unique 3D fold Base triplet interaction involving ribose zipper Base triplet interaction involving ribose zipper C75 projects deep into the core of the ribozyme Perturbation of pKa for C75 by surrounded hydrogen-bonding network Perturbation of pKa under microenvironment created by RNA tertiary fold. The Adenosine and Cytosine could act as general base The Histidine rescue experiment on C76U mutant of HDV ribozyme An HDV-like sequence in the human CPEB3 gene Science 313, 1788-1792 (2006) Control of gene expression by a natural metabolite-responsive ribozyme Nature 428, 281-286 (2004) RNA cleavage by acid-base catalysis and two-metal ion catalysis Initially, metal ions were proposed to supply the chemical versatility that nucleotides lack. However, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly rely on essential metal-ion cofactors, self-cleaving ribozymes (HDV) seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences in chemical features, both RNA and protein enzymes use similar catalytic strategies. Control of gene expression by a natural metabolite-responsive ribozyme-glmS RNA ribozyme- • glmS RNA resides upstream from the glmS gene in B. subtilis and at least 17 other Gram-positive bacteria. • The glmS gene encodes the enzyme glutamine-fructose-6- phosphate amidotransferase, which uses fructose-6- phosphate (Fru6P) and glutamine to generate glucosamine-6 -phosphate (GlcN6P). This reaction is the first step in the pathway for the production of UDP-GlcNAc, which is subsequently used in the process of cell wall biosynthesis. Nature 428, 281-286 (2004) glmS RNA has been splited into sepatate substrate and ribozyme domains 5 nM 5΄-32P-labeled RNA substrate was incubated for 5 min at 23°C in the presence of 100 nM ribozyme and in the absence or presence of 100 mM effector as indicated for each experiment. glmS RNA responds to GlcN6P with exceptional molecular specificity A pre-organized active site Science 313, 1752-1756 (2006) Base-triple help organizing the roof of the active site The floor of the active site is formed by three connected loops A solvent-accessible activator- binding pocket A coenzyme-binding pocket floor A general base catalysis provided by 2-amine of GlcN6P Autoregulation of GlmS enzyme mediated by metabolite-induced ribozyme cleavage of mRNA Comparison of side chain diversity in protein and RNA Peptidyl-transferase Is Ribosome a ribozyme? The peptidyl transferase center is located on the large subunit.
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