上海交通大学 生物技术与人类论文 第二次论文 INGHAI JIAO TONG UNIVERS 院（系)生命科学技术学院专业生物工程 姓名张馥雅 学号5100809068
生物技术与人类论文 第二次论文 院（系）生命科学技术学院 专业 生物工程 姓名 张馥雅 学号 5100809068
The prospects and inquiry of ribosome function and structure Zhang Fuya School of Life Sciences and Biotechnology Shanghai Jiao Tong University,Shanghai 200240,China Abstract Ribosome is a ribozyme,which found by screening technology in vitro is the same as ribosomes and can catalytic the formation of peptide bond.RNA plays the important role in the origin of life. With the discovery of RNA multi-function,RNA is regarded as the bio macromolecule of life and gets more attention and application in the new drug research. Keywords:RNA:Ribozyme:Peptidyl transferase 1.Introduction There are three hypotheses about rRNA Atomic resolution crystal structures of the function:1.rRNA is mainly acted as the large subunit published since the middle of structural skeleton of ribosomal protein August 2000 prove that the peptidyl transfe- assembly.The ribosomal protein takes rase center of the ribosome,which is the site catalytic effect in protein synthesis 2.rRNA is of peptide-bond formation,is composed a kind of substance that determines the protein entirely of RNA[1];the ribosome is a sequence 3.With its catalytic action,rRNA ribozyme.They also demonstrate that directly catalyzes protein synthesis.[2] alignment of the CCA ends of ribosome-bound peptidyl tRNA and 3.Ribosome is a kind of ribozyme aminoacyl tRNA in the peptidyl transfe- In 1982,T.Cech proved that RNA has rase center contributes significantly to its catalytic function by researching tetrahymena, catalytic power. a kind of protozoa,and he called it ribozyme. Since then,the catalytic function of RNA has 2.Some hypotheses been found in nature constantly.T.Cech and Protein synthesis is the most complicated S.A Itman also won the 1989 Nobel Prize for and core process of cell metabolism,which chemistry because of the discovery of involves more than 200 kinds of ribozyme.[3]The discovery of ribozyme has biomacromolecule take part in.Protein an important meaning,which makes people processing place-ribosome is a complex realize that RNA's biological function is not which made of the ribosomal RNA(rRNA) simply transferring genetic information,and and ribosomal protein.The content of Protein people begin to look again at the biological is about a third,and the content of rRNA is function of RNA.Recently,through the about two-thirds.In the protein biosynthesis, analysis of X-ray diffraction about the whether rRNA or protein play a leading role crystallization of ribosomal large and small has always been interested by people and there subunit,scientists confirm the peptide bond is are many hypotheses have been put forward. formed by rRNA catalysis and the ribosome is
The prospects and inquiry of ribosome function and structure Zhang Fuya School of Life Sciences and Biotechnology Shanghai Jiao Tong University, Shanghai 200240, China Abstract Ribosome is a ribozyme, which found by screening technology in vitro is the same as ribosomes and can catalytic the formation of peptide bond. RNA plays the important role in the origin of life. With the discovery of RNA multi-function, RNA is regarded as the bio macromolecule of life and gets more attention and application in the new drug research. Keywords: RNA; Ribozyme; Peptidyl transferase 1. Introduction Atomic resolution crystal structures of the large subunit published since the middle of August 2000 prove that the peptidyl transferase center of the ribosome, which is the site of peptide-bond formation, is composed entirely of RNA[1]; the ribosome is a ribozyme. They also demonstrate that alignment of the CCA ends of ribosome-bound peptidyl tRNA and aminoacyl tRNA in the peptidyl transferase center contributes significantly to its catalytic power. 2. Some hypotheses Protein synthesis is the most complicated and core process of cell metabolism, which involves more than 200 kinds of biomacromolecule take part in. Protein processing place——ribosome is a complex which made of the ribosomal RNA (rRNA) and ribosomal protein. The content of Protein is about a third, and the content of rRNA is about two-thirds. In the protein biosynthesis, whether rRNA or protein play a leading role has always been interested by people and there are many hypotheses have been put forward. There are three hypotheses about rRNA function: 1. rRNA is mainly acted as the structural skeleton of ribosomal protein assembly. The ribosomal protein takes catalytic effect in protein synthesis 2. rRNA is a kind of substance that determines the protein sequence 3. With its catalytic action, rRNA directly catalyzes protein synthesis. [2] 3. Ribosome is a kind of ribozyme In 1982, T.Cech proved that RNA has catalytic function by researching tetrahymena, a kind of protozoa, and he called it ribozyme. Since then, the catalytic function of RNA has been found in nature constantly. T.Cech and S.A Itman also won the 1989 Nobel Prize for chemistry because of the discovery of ribozyme. [3]The discovery of ribozyme has an important meaning, which makes people realize that RNA’s biological function is not simply transferring genetic information, and people begin to look again at the biological function of RNA. Recently, through the analysis of X-ray diffraction about the crystallization of ribosomal large and small subunit, scientists confirm the peptide bond is formed by rRNA catalysis and the ribosome is
a kind of ribozyme.We can come to the tRNA of P site and the aminoacyl-tRNA of A conclusion that the ribosomal protein take a site,which is called peptidyl transferase center mainly pole in maintaining the conforma- (Traut and Monro 1964;Monro 1967)[4]. tion of rRNA,a supplementary role and in Within the position of peptide bond formation protein synthesis process,rRNA plays a very at the range of 2 nm,there is no protein important role electron cloud.Peptidyl transferase center is completely consist of 23S rRNA domain,and 4.Peptidyl transferase center ribosomal protein take a mainly pole in The fine structure of ribosomal large maintaining the conformation of rRNA.a subunit shows that the bottom of ribosomal supplementary role. large subunit's cavity,is the position which made by the interaction between the peptidyl (A) L18 (B) L21e 10e L6 Fig.1.Crystal structure of Ribosomes peptidyl transferase reaction center[2] (A)rRNA peptidyl transferase reaction center:purple is peptidyl transferase reaction center,red line is RNA,other colors band is protein. (B)The nearest protein from the peptidyl transferase reaction center:purple line is polypeptide;purple dot is peptidyl transferase reaction center 5.Ribosome peptidyl transferase reaction the stability of superoxide anion's transition mechanism state,etc.Biochemical research data indicate Although it has already confirmed that that the synthesis of peptide bond is a partial peptidyl transferase reaction center of, entropy-driven reaction,not a enthalpy-driven rRNA nucleoside take an important part in reaction[5].Based on this,some researchers catalytic,but its mechanism of action is not have speculated that in the reaction of peptide very clear.Currently,there are several bond formation,reactants and the surrounding hypotheses about the ribosome catalytic H2O molecular interact to form a hydrogen- reaction mechanism,such as entropy-driven bond network,which is greatly reduces the Reaction,proton shuttle mechanism,through free energy of activation in the ribosome
a kind of ribozyme. We can come to the conclusion that the ribosomal protein take a mainly pole in maintaining the conformation of rRNA, a supplementary role and in protein synthesis process, rRNA plays a very important role. 4. Peptidyl transferase center The fine structure of ribosomal large subunit shows that the bottom of ribosomal large subunit’s cavity, is the position which made by the interaction between the peptidyl tRNA of P site and the aminoacyl-tRNA of A site, which is called peptidyl transferase center (Traut and Monro 1964; Monro 1967) [4]. Within the position of peptide bond formation at the range of 2 nm, there is no protein electron cloud. Peptidyl transferase center is completely consist of 23S rRNA domain, and ribosomal protein take a mainly pole in maintaining the conformation of rRNA, a supplementary role. Fig.1.Crystal structure of Ribosomes peptidyl transferase reaction center[2] (A) rRNA peptidyl transferase reaction center: purple is peptidyl transferase reaction center, red line is RNA, other colors band is protein. (B)The nearest protein from the peptidyl transferase reaction center: purple line is polypeptide; purple dot is peptidyl transferase reaction center 5. Ribosome peptidyl transferase reaction mechanism Although it has already confirmed that partial peptidyl transferase reaction center of, rRNA nucleoside take an important part in catalytic, but its mechanism of action is not very clear. Currently, there are several hypotheses about the ribosome catalytic reaction mechanism, such as entropy-driven Reaction, proton shuttle mechanism, through the stability of superoxide anion’s transition state, etc. Biochemical research data indicate that the synthesis of peptide bond is a entropy-driven reaction, not a enthalpy-driven reaction[5]. Based on this, some researchers have speculated that in the reaction of peptide bond formation, reactants and the surrounding 𝐻2𝑂 molecular interact to form a hydrogenbond network, which is greatly reduces the free energy of activation in the ribosome
catalytic peptide bond synthetic reaction[6]. [7].The hydrogen bonds these groups form There are,however,three groups in the with the a-amino group,either singly or neighborhood of the reactive a-amino group collectively,almost certainly help align it,and that could conceivably form hydrogen bonds if any of these groups were to have an with it,and two are likely to do so:(1)the elevated PKa,its hydrogen-bonding 2'-OH of A76 in the P-site bound peptidyl interaction would facilitate the tRNA,(2)the N3 of A2486(A2451in peptide-bond-forming reaction by enhancing Escherichia coli)of 23S rRNA,and (3)the the nucleophilicity of the a-amino group[8]. 2'-OH of A2486(2451;Hansen et al.2002) A76 Phe C75 attacking amino N3 Tyr A2486 (2451)） Fig.2.A model for the peptidyl transferase center of the large ribosomal subunit from Haloarcula marismortui with substrates bound to both the A site and its P site.This model was obtained by superimposing the structure of an A-site substrate complex (PDB#IFGO)on the structure of a P-site substrate complex(PDB #1M90:Hansen et al.2002).The a-amino group of the A-site substrate(purple)is positioned for a pro-S attack on the carbonyl carbon of the ester linking the peptide moiety of the P-site substrate(green).Possible hydrogen-bonding interactions involving the a-amino group and the N3 of A2486(2451)and the 2'-OH of A76 are indicated.The 2'-OH of A2486(2451)is also close enough so that it could interact.[7] Although the ribosome catalytic reaction substrate and nucleophile a-amino group mechanism is still not very clear,the 2'-OH of interact with 23S rRNA base which is located A76 which belongs to the P site of tRNA plays in the active center.The lowest-molecular an important role has been made clear[9]. -weight aminoacyl tRNA-like substrate the Through the CCA sequence of A and P site ribosome will accept is puromycin,which is
catalytic peptide bond synthetic reaction[6]. There are, however, three groups in the neighborhood of the reactive -amino group that could conceivably form hydrogen bonds with it, and two are likely to do so: (1) the 2’-OH of A76 in the P-site bound peptidyl tRNA, (2) the N3 of A2486 (A2451in Escherichia coli) of 23S rRNA, and (3) the 2’-OH of A2486 (2451; Hansen et al. 2002) [7]. The hydrogen bonds these groups form with the -amino group, either singly or collectively, almost certainly help align it, and if any of these groups were to have an elevated , its hydrogen-bonding interaction would facilitate the peptide-bond-forming reaction by enhancing the nucleophilicity of the -amino group[8]. Fig.2. A model for the peptidyl transferase center of the large ribosomal subunit from Haloarcula marismortui with substrates bound to both the A site and its P site. This model was obtained by superimposing the structure of an A-site substrate complex (PDB#1FGO) on the structure of a P-site substrate complex (PDB #1M90; Hansen et al. 2002). The -amino group of the A-site substrate(purple) is positioned for a pro-S attack on the carbonyl carbon of the ester linking the peptide moiety of the P-site substrate (green). Possible hydrogen-bonding interactions involving the -amino group and the N3 of A2486 (2451) and the 2’-OH of A76 are indicated. The 2’-OH of A2486 (2451) is also close enough so that it could interact.[7] Although the ribosome catalytic reaction mechanism is still not very clear, the 2′-OH of A76 which belongs to the P site of tRNA plays an important role has been made clear[9]. Through the CCA sequence of A and P site substrate and nucleophile -amino group interact with 23S rRNA base which is located in the active center. The lowest-molecular -weight aminoacyl tRNA-like substrate the ribosome will accept is puromycin, which is
effectively an adenosine aminoacylated with significant rearrangement of the conformation tyrosine,and molecules as small as CCA of the phosphate from that seen when aminoacylated with N-formylmethionine will CCdApPmn is bound in the ribosomal active serve in place of peptidyl tRNA.Because the site.Such structures will be more relevant to peptidyl transferase center is entirely the reality of peptidyl transferase catalysis by contained in the large subunit,it should be the ribosome.In addition to allowing inclusion possible to learn a lot about the mechanism of of the critical A76 2'-OH,our synthetic peptide-bond formation from crystal structures scheme imparts modularity to the production of large subunits with substrates like these of inhibitors,allowing the synthesis of a large bound this A and P site substrate exactly variety of related molecules.It particularly arranges in the active center.The proton affords flexibility in the tetrahedral phosphate shuttles which react at the 2'-OH of A76 and P-site portions of the molecules.Among which belongs to the P site of tRNA,forming other changes,we will be able to synthesize 6-membered ring transition state is considered transition state analogs with chiral centers at the most advantageous catalytic pathway,and their tetrahedral phosphates.This will make it the reaction does not involve the chemical possible to resolve the stereo chemical catalysis of ribosomal RNA group[10].There ambiguity of the peptidyl transferase transition are substantial steric constraints placed on the state.[11]Interactions involving the 2-OH of tetrahedral phosphate by the now present A76 could help position the attacking amino 2'-OH of A76.Therefore,we expect a Substrates Transition state P-site A-site 0 H aa -peptide OH Products H P-site A-site OH OH 0=C OH NH 0= aa peptide
effectively an adenosine aminoacylated with tyrosine, and molecules as small as CCA aminoacylated with N-formylmethionine will serve in place of peptidyl tRNA. Because the peptidyl transferase center is entirely contained in the large subunit, it should be possible to learn a lot about the mechanism of peptide-bond formation from crystal structures of large subunits with substrates like these bound this A and P site substrate exactly arranges in the active center. The proton shuttles which react at the 2′-OH of A76 which belongs to the P site of tRNA, forming 6-membered ring transition state is considered the most advantageous catalytic pathway, and the reaction does not involve the chemical catalysis of ribosomal RNA group[10]. There are substantial steric constraints placed on the tetrahedral phosphate by the now present 2'-OH of A76.Therefore, we expect a significant rearrangement of the conformation of the phosphate from that seen when CCdApPmn is bound in the ribosomal active site. Such structures will be more relevant to the reality of peptidyl transferase catalysis by the ribosome. In addition to allowing inclusion of the critical A76 2'-OH, our synthetic scheme imparts modularity to the production of inhibitors, allowing the synthesis of a large variety of related molecules. It particularly affords flexibility in the tetrahedral phosphate and P-site portions of the molecules. Among other changes, we will be able to synthesize transition state analogs with chiral centers at their tetrahedral phosphates. This will make it possible to resolve the stereo chemical ambiguity of the peptidyl transferase transition state. [11]Interactions involving the 2_-OH of A76 could help position the attacking amino