Ribozymes, types of ribozymes.

A ribozyme is a ribonucleic acid (RNA) enzyme that catalyzes a chemical reaction. The ribozyme catalyses specific reactions in a similar way to that of protein enzymes. Also called catalytic RNA, ribozymes are found in the ribosome where they join amino acids together to form protein chains.

1. ribozymes JAYDIP D. PARADAVA J&J COLLEGE OF SCIENCE ROLL NO. 4112 MSc. MICROBIOLOGY

2. What is ribozyme ? A ribozyme (ribonucleic acid enzyme) is an RNA molecule that is capable of performing specific biochemical reactions, similar to the action of protein enzymes.

3. history 1967: Carl Woese , Francis Crick , and Leslie Orgel were the first to suggest that RNA could act as a catalyst. 1970s: Thomas Cech, at the University of Colorado , was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for splicing reaction, he found that intron could be spliced out in the absence of any added cell extract . As much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break and reform phosphodiester bonds.

4. Sidney Altman, a professor at Yale University , was studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme called RNase-P , which is responsible for conversion of a precursor tRNA into the active tRNA. Much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme.  1981-82: Discovery of Ribozyme  1982: Ribozyme term was introduced by Kelly Kruger et al. in a paper published in The Cell  1989: Thomas Cech and Sidney Altman shared the Nobel Prize fo demonstrating that RNA could act as an enzyme.

5. Types of ribozymes Group I and group II intron splicing ribozymes RNase P Hammerhead Ribozyme Hairpin ribozyme Ribosome

6. Group 1 intron splicing Group I intron ribozymes constitute one of the main classes of ribozymes.  Found in bacteria, lower eukaryotes and higher plants.  Group I introns are also found inserted into genes of a wide variety of bacteriophages of Gram-positive bacteria.  However, their distribution in the phage of Gram-negative bacteria is mainly limited to the T4, T-even and T7-like like bacteriophages. The group I splicing reaction requires a guanine residue cofactor, the 3’ OH group of guanosine is used as a nucleophile. The 3’ OH group attacks the 5’ phosphate of the intron and a new phosphodiester bond is formed. The 3’ OH of the exon that is displaced now acts as the nucleophile in a similar reaction at the 3’ end of the intron. So the intron is precisely excised and exons are joined together.

7. Group 2 intron splicing Group II introns have been found in bacteria and in the mitochondrial and chloroplast genomes of fungi, plants, protists, and an annelid worm. Mechanism: The 2’OH of a specific adenosine acts as a nucleophile and attacks the 5’ splice site creating a branched intron structure. The 3’ OH of the 5’ exon attacks the 3’ splice site, ligating the exons and releasing the intron as a lariat structure.

8. Rnase P : Ribonuclease P (RNaseP), a ribonucleoprotein, is an essential tRNA processing enzyme found in all living organisms. Mechanism: • All RNase P enzymes are ribonucleoproteins [bacteria: 1RNA + 1 protein subunit; eukaryotes: 1 RNA + many protein subunits (11 in human)], • In Ribonuclease – P, protein component is facilitates binding between RNase and t-RNA substrate. • Requires divalent metal ions (like Mg2+) for its activity. • Endo-ribonuclease responsible for generating 5’ end of matured tRNA molecules. • Cleavage via nucleophilic attack on the phosphodiester bond leaving a 5’-phosphate and 3’-hydroxyl at the cleavage site.

9. Hammerhead ribozyme Hammerhead ribozymes (HHRZs) are tiny autocatalytic RNAs that cleave single-stranded RNA. They are found in nature as a part of certain virus-like elements called virusoids , which use a "rolling-circle replication" mechanism to reproduce their small, circular RNA genomes. • The HHRZ is so named because its secondary structure is similar to that of a hammer head, but actually its tertiary structure is more like ‘Y’ shaped. Rolling-circle replication initially produces a long strand of multiple copies of the virusoid RNA. Each copy contains a hammerhead motif that catalyzes strand breakage between itself and the next copy in the transcript. Thus, by virtue of HHRZ motifs, the long strand breaks itself into many individual molecules.

10. Hairpin ribozyme The hairpin ribozyme is an RNA motif that catalyzes RNA processing reactions essential for replication of the satellite RNA molecules in which it is embedded. These reactions are self processing, i.e. a molecule rearranging its own structure. Both cleavage and end joining reactions are mediated by the ribozyme motif. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens.

11. Ribosomes Ribosome is a large and complex molecular machine, found within all living cells, that serves as the primary site of biological protein synthesis (translation). It consists of two sub-units, one large and one small. The large(50s) subunit has 5s and 23s rRNAs as its core. After the determination of the high-resolution structure of ribosome, it was clear that the 23s subunit is responsible for the catalytic peptidyl transferase activity that links amino acids together. That is why ribosome is also a ribozyme.  The red region indicates the site where mRNA interacts with the tRNA anticodons.

12. ribo switch

13. Genetic regulation by RNA is widespread in bacteria. One common form of riboregulation in bacteria is the use of ribonucleic acid sequences encoded within mRNA that directly affect the expression of genes encoded in the full transcript (called cis-acting elements because they act on the same molecule they're coded in). These regulatory elements are known as riboswitches and are defined as mRNA elements that bind metabolites or metal ions as ligands and regulate mRNA expression by forming alternative structures in response to this ligand binding.

14. Most known riboswitches occur in bacteria, but functional riboswitches of one type (the TPP riboswitch) have been discovered in plants and certain fungi.

15. Mechanism :  Riboswitch-mediated changes in gene expression can occur either transcriptionally or translationally . The expression platform for a riboswitch that acts during transcription typically involves the ligand-dependent formation of an intrinsic terminator or anti-terminate structure.

16. application  Riboswitches as tools for regulated gene expression  Ligand-inducible expression systems are important genetic tools for common laboratory organisms such as E. coli and B. subtilis. However, inducers (such as IPTG) are too expensive. Natural riboswitches that are activated by amino acids may therefore represent an affordable alternative for such applications. Toward this goal, a tandem glycine riboswitch from B. subtilis was used for glycine inducible production of β-galactosidase in B. subtilis cells.  Just as natural riboswitches can regulate gene expression in response to small-molecule ligands during transcription or translation , synthetic riboswitches can be engineered to repress or activate any gene expression in a ligand-dependent fashion. This feature should enable RNA switches to play an increasingly important role as chemical biologists seek to modulate many types of cellular behavior in response to a broad range of chemical signals.

17. REFERENCE: Lehninger Principles of Biochemistry SIXTH EDITION David L. Nelson Professor of Biochemistry University of Wisconsin–Madison Michael M. Cox Professor of Biochemistry University of Wisconsin–Madison