分子遗传学课件转录及转录后修饰20150921宣讲培训.ppt

Where is the catalytic activity in RNase P? RNase P is composed of a 375 nucleotide RNA and a 20 kDa protein. The protein component will NOT catalyze cleavage on its own. The RNA WILL catalyze cleavage by itself !!!! The protein component aids in the reaction but is not required for catalysis. Thus RNA can be an enzyme. Enzymes composed of RNA are called ribozymes. Hammerhead ribozymes A 58 nt structure is used in self-cleavage The sequence CUGA adjacent to stem-loops is sufficient for cleavage Mechanism 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 rRNA), phage Eukaryotes lower (algae, protists, fungi) nuclear rRNA genes, organellar genes, Chlorella viruses higher plants: organellar genes lower animals (Anthozoans): mitochondrial 1800 known, classified into ~12 subgroups, based on secondary structure Splicing of pre-mRNA 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 complexity 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 pre-mRNA 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 nuclea