三联体密码（课件

,The genetic code and tRNA,The genetic code tRNA structure and function,The genetic code,Nature Deciphering Features Effect of mutation ORFs Overlapping genes,Nature,The genetic code (triplet codon) The genetic code is the correspondence between the sequence of the four bases in nucleic acids and the sequence of the 20 amino acids in proteins. Triplets: 43=64, amino acids: 20 (degenerate) The genetic code is very nearly, but no quite, universal. The rule of triplet coden,三联体密码(triplet coden),mRNA分子上从5至3方向，由AUG开始，每3个核苷酸为一组，决定肽链上某一个氨基酸或蛋白质合成的起始、终止信号，称为三联体密码 起始密码(initiation coden): AUG 终止密码(termination coden): UAA，UAG，UGA 三联体密码以线性方式阅读，中间不重叠也不停顿,遗传密码表,密码子使用规律,原核生物中大部分以AUG为起始密码子，少数使用GUG，真核生物则全部使用AUG为起始密码子，而终止密码子UAA、UAG、UGA全部被使用，有时连用两个密码子， 以便更保险地终止肽链合成。 哺乳动物中，对同义密码子的使用差异甚大，有的广泛使用，有些则有所偏爱。 大肠杆菌中，以C、U为结尾的同义密码，若前两个碱基为A、U，则第三位碱基优先使用C而不是U，如前两个碱基为C、G，则优先使用U。,Deciphering,1960s, Nirenberg, E.coli , cell-free protein synthesizing system,Features,Directivity Continuity Degeneracy Universality Wobble,连续性(continuity),编码蛋白质氨基酸序列的各个三联体密码连续阅读，密码间既无间断也无交叉。 基因损伤引起mRNA阅读框架内的碱基发生插入或缺失，可能导致框移突变(frameshift mutation)。,Degeneracy,Only methionine and tryptophan have single codons. Generally they differ only in their third position. In all cases, if the third position is a pyrimidine, then the codons specify the same amino acid (are synonymous). In most cases, if the third position is a purine the codons are also synonymous. If the second position is a pyrimidine then generally the amino acid specified is hydrophilic. If the second position is a purine then generally the amino acid specified is polar.,遗传密码表,通用性(universality),蛋白质生物合成的整套密码，从原核生物到人类都通用。 少数例外，如动物细胞的线粒体、植物细胞的叶绿体。,摆动性(wobble),转运氨基酸的tRNA的反密码需要通过碱基互补与mRNA上的遗传密码反向配对结合，但反密码与密码间不严格遵守常见的碱基配对规律，称为摆动配对。,U,摆动配对,密码子、反密码子配对的摆动现象,Effect of mutation,Mutation types: transition, transversion In the third position, transitions usually have no effect, but can cause changes between Met and Ile, or Trp and stop. Just over half of transversions in the third position have no effect and the remainder usually result in a similar type of amino acid being specified, for example Asp or Glu.,遗传密码表,In the second position, transitions will usually result in a similar chemical type of amino acid being used, but transversions will change the type of amino acid. In the first position, mutations (both transitions and transversions) usually specify a similar type of amino acid, and in a few cases it is the same amino acid.,ORFs,从mRNA 5端起始密码子AUG到3端终止密码子之间的核苷酸序列，各个三联体密码连续排列编码一个蛋白质多肽链，称为开放阅读框架(open reading frame, ORF)。 An ORF is a suspected coding region.,Overlapping genes,Although it is generally true that one gene encodes one polypeptide, and the evolutionary constraints on having more than one protein encoded in a given region of sequence are great, there are now known to be several examples of overlapping coding regions (overlapping genes). Generally these occur where the genome size is small and there is a need for greater information storage density.,e.g. Phage X174,Genome:5386bp Protein: 11, 262kDa. Without overlapping genes, this genome could encode at most 200kDa of protein. Three proteins are encoded within the coding regions for longer proteins.,In prokaryotes, the ribosomes simply have to find the second start codon to be able to translate the overlapping gene and they may achieve this without detaching from the gemplate. Eukaryotes have a different way of initiating protein synthesis and tend to make use of alternative RNA processing to generate variant proteins from one gene.,tRNA structure and function,tRNA structure tRNA function Aminoacylation of tRNAs Aminoacyl-tRNA synthetases Proofreading,tRNA structure,tRNA primary structure tRNA secondary structure tRNA tertiary structure,tRNA primary structure,tRNAs are the adaptor molecules that deliver amino acids to the ribosome and decode the information in mRNA. Their primary structure is 50-95nt long. (common: 76) They have many modified bases. (sometimes: 20%) 50 different types of modification, several hundred tRNA molecules, post-transcriptional modification In the tRNA primary structure, there are 15 invariant nucleotides and eight which are either purines (R) or pyrimidines (Y).,Using the standard numbering convention where position 1 is the 5-end and 76 is the 3-end. D-loop: 14-21 Anticodon loop: 32-37 T-loop: 53-61 Acceptor: 74-76,tRNA secondary structure,Almost all tRNAs have a common secondary structure (i.e. base pairing of different regions to form stems and loops). Exception: Yeast Some Mit tRNA,This structure has a 5-phosphate formed by RNase P cleavage, not the 5-triphosphate. It has a 7 bp stem formed by base pairing between the 5- and 3-ends of the tRNA; however, the invariant residues 74-76 (i.e. the terminal 5-CCA-3) which are added during processing in eukaryotes are not included in this base pairing region.,Amino acid acceptor stem,D-arm,Working 5- to 3 (anticlockwise), the next secondary structural feature is called the D-arm which is composed of a 3 or 4 bp stem and a loop called the D-loop (DHU-loop) usually containing the modified base dihydrouracil.,Anticodon loop,The next structural feature consists of a 5 bp stem and a seven residue loop in which there are three adjacent nucleotides called the anticodon which are complementary to the codon sequence (a triplet in the mRNA) that the tRNA recognizes.,Variable arm,It can have between three and 21 residues and may form a stem of up to 7 bp. The other positions of length variation in tRNAs are in the D-loop.,TC-arm,T arm It is composed of a 5 bp stem ending in a loop containing the invariant residues GTC-arm.,Majority of the invariant residues in tRNA molecules are in the loops and do not play a major role in forming the secondary structure. Several of them help to form the tertiary structure.,tRNA tertiary structure,There are nine hydrogen bonds (tertiary hydrogen bonds) that help form the 3-D structure of tRNA molecules. They mainly involve base pairing between several invariant bases. Base pairing between residues in the D- and T-arms fold the tRNA molecule over into an L-shape, with the anticodon at one end and the amino acid acceptor site at the other. The tRNA tertiary structure is strengthened by base stacking interactions.,tRNA的三级结构示意图,tRNA function,tRNAs are joined to amino acids to become aminoacyl-tRNAs (charged tRNAs) in a reaction called aminoacylation. It is these charged tRNAs that are the adaptor molecules in protein synthesis. Special enzymes called aminoacyl-tRNA synthetases carry out the joining reaction which is extremely specific. These pairs of specific amino acids and tRNAs, or tRNAs and aminoacyl-tRNA synthetases are called cognate pairs.,tRNA是一个万能接头,对氨酰- tRNA合成酶的识别位点（接头合成酶） 3端-CCA上的氨基酸运载位点（接头氨基酸，装载） 对核糖体的识别位点（将氨基酸运送到目的地） 反密码子位点（接头mRNA，验货并卸载）,tRNA是联系mRNA和蛋白质的适配器,tRNA在蛋白质合成过程中的作用至关重要,每一种氨基酸在被掺入肽链之前都必须首先被活化和连接在专一tRNA上 载体tRNA凭借自身的反密码子与mRNA上的密码子相识别而把所携带的氨基酸送到肽链的一定位置上 遗传信息是通过mRNA上的密码子与tRNA上的反密码子间碱基配对作用翻译出来的 。,Aminoacylation of tRNAs,游离氨基酸在掺入肽链以前必须活化以获得额外的能量: 活化：游离氨基酸在专一的氨酰tRNA合成酶的帮助下形成中间复合物。 连接：中间复合物与专一的tRNA相连，tRNA负责将它带到核糖体上的特定位点（A位点上）并添加到正在合成的肽链C末端。 氨酰-tRNA的表示方法,活化,在Mg2+的存在下，氨酰tRNA合成酶识别并结合专一的配体氨基酸，然后氨基酸的羧基与细胞环境中的ATP发生反应形成一个酸酐型的高能复合物（氨酰AMP中间复合物）。该中间复合物暂时结合在酶上。 氨基酸 + ATP 氨酰AMP-酶 + PPI,第一步反应,氨基酸 ATP-E 氨酰-AMP-E PPi,连接,氨酰tRNA合成酶上还存在专一的tRNA识别位点，特定的游离tRNA会识别并结合到氨酰AMP-酶复合物的活性部位，此时氨基酸就会被转移到tRNA的3端，其羧基与tRNA 3端的自由-OH形成氨酰酯键，从而形成氨酰tRNA。 氨酰AMP-酶 + tRNA 氨酰tRNA + AMP + 酶,第二步反应,氨酰-AMP-E tRNA 氨酰-tRNA AMP E,氨酰tRNA形成后，由tRNA来决定进一步的去向，tRNA凭借自身的反密码子与mRNA上的密码子相识别，把所携带的氨基酸送到肽链的 一定位置上。每一个密码子对应的肽链位置上都能掺入正确的氨基酸。,氨酰-tRNA的表示方法：,Ala-tRNAAla Ser-tRNASer Met-tRNAMet,Aminoacyl-tRNA synthetases,Despite the fact that they all carry out the same reaction of joining an amino acid to a tRNA, the various synthetase enzymes can be quite different. They fall into one of four classes of subunit structure, being either , 2,4 or 22. The polypeptide chains range from 334 to over 1000 amino acids in length, and these enzymes contact the tRNA on the underside (in the angle) of the L-shape. They have a separate amino acid-binding site.,The synthetases