生物化学原理课件（英文）：Chapter11 Structure and function of ribozymes

1、Chapter 11 Structure and Function of Ribozymes Outline Definition and Discovery of Ribozymes Comparison between Protein-based and RNA- based Enzymes Types of ribozymes 1.Small ribozymes 2.Large ribozymes Mechanism of Ribozyme Catalysis 1.Mechanism of small ribozyme catalysis 2.Mechanism of large rib

2、ozyme catalysis Significance of the discovery of ribozymes and its applications (self-study) Discovered by Tom Cech and Sydney Altman (the Nobel prize laureates) Ribozymes just are catalytic RNA molecules, which can catalyze a number of the chemical reactions Ribozymes can be naturally-occurring or

3、artificially-made Two naturally-occurring ribozymes are present in almost every organism: RNase P and Ribosome Cech Altman Discovery and Definition of Ribozymes 1.ribozyme rate enhancements can be substantial. For example, the rate constant for chemistry of the self-cleaving HDV ribozyme is estimate

4、d at 102104 s1, which is close to the maximal cleavage rate of RNase A. 2.ribozymes can use cofactors during catalysis, and they can be switched on and off by the binding of small-molecule allosteric effectors. 3.ribozymes, like protein catalysts, fold into specific 3D shapes that can harbor deep gr

5、ooves and solvent-inaccessible active sites. These tertiary structures facilitate catalysis in part by orienting substrates adjacent to catalytic groups and metal ions. To facilitate chemical transformations, catalysts stabilize the transition state. Both protein and RNA catalysts may achieve this b

6、y adding or removing protons during a reaction, orienting substrates so that they are optimally positioned to react, and using binding interactions away from the reaction site to force an unfavorable contact that is relieved in the transition state. Common points between Protein-based and RNA-based

7、Enzymes 1.Proteins and RNA are very different polymers. The phosphodiester backbone of nucleic acids is charged, and thus RNA has a large electrostatic component in which metal ions have a big role to play. 2.The range of chemical reactions that are catalyzed by ribozymes is rather limited, and the

8、rate enhancements are relatively modest because the range of functional groups that can be called upon to participate in the chemistry is limited compared to the set of amino acid side-chains. 3.The great majority of reactions carried out by the ribozymes are transesterification reactions, although

9、other reactions can be catalyzed in a few cases . Differences between Protein-based and RNA-based Enzymes Naturally occurring ribozymes - are classified into two different groups, the large and small ribozymes, based on differences in size and reaction mechanism. Artificial ribozymes - produced by i

10、n vitro selection Types of Ribozymes Small Ribozymes and Large Ribozymes Small ribozymes includes the hammerhead and hairpin motif, the hepatitis delta virus (HDV) RNA, the Varkud satellite (VS) RNA, and the glmS riboswitch ribozyme. These ribozymes range in size from about 40 -154 nt. The hammerhea

11、d, so called for its three helices in a T shape, is the most frequently found catalytic motif in plant pathogenic RNAs, such as viroids. The hairpin ribozyme has only been found in some virusoids. The HDV RNA is a viroid-like satellite virus of the HBV. The VS ribozyme is part of a larger RNA that i

12、s transcribed from a plasmid found in the mitochondria of some strains of Neurospora crassa. The glmS riboswitch ribozyme is involved in regulating bacterial gene expression Small ribozymes They vary in size from a few hundred nucleotides up to about 3000nt, and are further distinguished from the sm

13、all ribozymes by all cleaving RNA to generate 3-OH termini, as opposed to a product with a 2,3-cyclic phosphate and a product with a 5-OH terminus. They include RNA component of RNase P, self-splicing group I and group II introns ,the RNA components of the spliceosome and the rRNAs catalyzing peptid

14、e bond formation during translation Large ribozymes Disadvantages of Ribozyme catalysis L RNA has a small repertoire of functional groups and these are embedded in a poorly constrained ribosyl-phosphate backbone heavy with negative charge. The pKas of the bases would appear to be either too low (3.5

15、 for A and 4.2 for C) or too high (9.8 for G and 10.5 for U) for use in efficient general acid catalysis or base catalysis. As a group, ribozymes, are less efficient catalysts relative to chemically supercharged proteins, yet in many cases ribozymes provide enough rate enhancement to have escaped re

16、placement by protein alternatives in the evolution from an RNA World to the protein dominated world of modern biochemistry. The catalytic mechanisms employed by RNA are chemically more diverse than initially suspected. Divalent metal ions, nucleobases, ribosyl hydroxyl groups, and even functional gr

17、oups on metabolic cofactors all contribute to the various strategies employed by Ribozymes. The lack of diverse functional groups in RNA molecules and the propensity for RNA to bind metal ions led to early hypotheses that all ribozymes might act as metalloenzymes, positioning metal ions for direct r

18、oles in catalysis. This seems to be true for group I and group II introns and RNase P. However, those ribozymes that perform site-specific strand scission the hammerhead, hairpin, VS and HDV ribozymes may use diverse catalytic mechanisms, and none has emerged clearly as a metalloenzyme. Mechanism of

19、 Ribozyme Catalysis Mechanism of small ribozyme catalysis Small ribozymes perform site-specific self-cleavage. Found in viral, virusoid, or satellite RNA genomes, they process the products of rolling circle replication into genome-length strands . The general mechanism of these ribozymes is similar

20、to that of many protein ribonucleases in which a 2 oxygen nucleophile attacks the adjacent phosphate in the RNA backbone, resulting in cleavage products with 2,3-cyclic phosphate and 5- OH Unlike protein ribonucleases, however, ribozymes cleave only at a specific location, using base-pairing and ter

21、tiary interactions to help align the cleavage site within the catalytic core. The evolutionary maintenance of these sequences maybe due to their site specificity and to the simplicity and efficiency of genome self-cleavage. Internal transesterification reaction catalyzed by the hammerhead, hairpin,

22、HDV, VS, and glmS ribozymes Small ribozymes perform site-specific self-cleavage Mechanism of large ribozyme catalysis In contrast to the small ribozymes, this class of ribozymes require divalent ions for them to function proficiently. Can be different from small ribozymes by all cleaving RNA to gene

23、rate 3-OH termini. A good example: Group I intron splicing promoted by two catalytic metal ions Large ribozymesSmall ribozymes Group I intron splicing promoted by two catalytic metal ions Natural catalytic RNAs are limited to reactions involving phosphoryl centers. As a result, a huge variety of art

24、ificial ribozymes have been selected by in vitro selection. This has yielded ribozymes using different catalytic metal ion cofactors. In addition to cleavage reactions, the catalyzed reactions include phosphorylation, ligation, polymerization and transesterification. They attackphosphoryl, carbonyl

25、or alkyl centers. Ribozymes with ribosyl-cleavage properties, a ligase activity (35, 25 and 55 phosphodiester linkage) and with a phosphorylation reaction could be selected. Other artificial ribozymes include selfaminoacylation, acyl transfer and aminoacyl transfer reactions, self-nitrogen and sulfu

26、r alkylation, biphenyl isomerization or porphyrin metalation. Even a ribozyme catalyzing a carboncarbon bond formation by the Diels-Alder reaction could be selected. Artificial Ribozymes The RNA world hypothesis proposes that self-replicating RNA molecules were precursors to current life, which is b

27、ased on DNA, RNA and proteins. RNA stores genetic information like DNA, and catalyzes chemical reactions like an enzyme protein. It may, therefore, have played a major step in the evolution of cellular life The RNA world would have eventually been replaced by the DNA, RNA and protein world of today, likely through an intermediate stage such as the