投影片 1 by yurtgc548

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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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