【文章內(nèi)容簡(jiǎn)介】 actions in the selfsplicing of Tetrahymena group I intron. Where is the catalytic activity in RNase P? RNase P is posed of a 375 nucleotide RNA and a 20 kDa protein. The protein ponent will NOT catalyze cleavage on its own. The RNA WILL catalyze cleavage by itself !!!! The protein ponent aids in the reaction but is not required for catalysis. Thus RNA can be an enzyme. Enzymes posed of RNA are called ribozymes. Hammerhead ribozymes ? A 58 nt structure is used in selfcleavage ? The sequence CUGA adjacent to stemloops is sufficient for cleavage CUGAGAC C G GG G C CAAAACUC GAGUCA C C A CU G G U GUB o n d th a t i s c l e a v e d .539。339。C U G A i s r e q u i r e d fo r c a ta l y s i sMechanism of hammerhead ribozyme ? The folded RNA forms an active site for binding a metal hydroxide ? Attracts a proton from the 2’ OH of the nucleotide at the cleavage site. ? This is now a nucleophile for attack on the 3’ phosphate and cleavage of the phosphodiester bond. 1989 Nobel Prize in chemistry, Sidney Altman, and Thomas Cech Distribution of Group I introns ? Prokaryotes – eubacteria (tRNA amp。 rRNA), phage ? Eukaryotes – lower (algae, protists, amp。 fungi) ? nuclear rRNA genes, anellar genes, Chlorella viruses – higher plants: anellar genes – lower animals (Anthozoans): mitochondrial ? 1800 known, classified into ~12 subgroups, based on secondary structure Splicing of premRNA ? The introns begin and end with almost invariant sequences: 5’ GU…AG 3’ ? Use ATP hydrolysis to assemble a large spliceosome (45S particle, 5 snRNAs and 65 proteins, same size and plexity as ribosome) ? Mechanism is similar to that of the Group II fungal introns: – Initiate splicing with an internal A – Uses a phosphoester transfer mechanism for splicing Initiation of phosphoester transfers in premRNA ? Uses 2’ OH of an A internal to the intron ? Forms a branch point by attacking the 5’ phosphate on the first nucleotide of the intron ? Forms a lariat structure in the intron ? Exons are joined and intron is excised as a lariat ? A debranching enzyme cleaves the lariat at the branch to generate a linear intron ? Linear intron is degraded Involvement of snRNAs and snRNPs ? snRNAs = small nuclear RNAs ? snRNPs = small nuclear ribonucleoproteins particles (snRNA plex with protein) ? Addition of these antibodies to an in vitro premRNA splicing reaction blocked splicing. ? Thus the snRNPs were implicated in splicing ? Recognizing the 5’ splice site and the branch site. ? Bringing those sites together. ? Catalyzing (or helping to catalyze) the RNA cleavage. Role of snRNPs in RNA splicing RNARNA, RNAprotein and proteinprotein interactions are all important during splicing snRNPs U1, U2, U4/U6, and U5 snRNPs – Have snRNA in each: U1, U2, U4/U6, U5 – Conserved from yeast to human – Assemble into spliceosome – Catalyze splicing Splicing of premRNA occurs in a “spliceosome” an RNAprotein plex premRNA spliced mRNA spliceosome (~100 proteins + 5 small RNAs) The spliceosome is a large proteinRNA plex in which splicing of premRNAs occurs. Assembly of spliceosome ? snRNPs are assembled progressively into the spliceosome. – U1 snRNP binds (and base pairs) to the 5’ splice site – BBP (branchpoint binding protein) binds to the branch site – U2 snRNP binds (and base pairs) to the branch point, BBP dissociates – U4U5U6 snRNP binds, and U1 snRNP dissociates – U4 snRNP dissociates ? Assembly requires ATP hydrolysis ? Assembly is aided by various auxiliary factors and splicing factors. Some RNARNA hybrids formed during the splicing reaction Steps of the spliceosomemediated splicing reaction A schematic diagram of six rearrangements that the spliceosome undergoes in mediating the first transesterification reaction in premRNA splicing. Assembly of spliceosome The spliceosome cycle The Significance of