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13.Translation and Proteins 13.1 Translation: Components Essential to Protein Synthesis • Ribosomal Structure • tRNA structure • Charging tRNA Ribosomal Structure • Because of its essential role in the expression of genetic information, the ribosome has been extensively analyzed. • One bacterial cell about 10,000 ribosome • One eukaryotic cell contains many times more. Electron microscopy reveals that the bacterial ribosome is about 250 um at its largest diameter two subunits, one large and one small. • Both subunits consist of one or more molecules of rRNA and an array of ribosomal proteins. The specific differences between prokaryotic and eukaryotic ribosomes are summarized in Figure 13-1 16S rRNA 小亚基30S 930kD 70S 2520kD 21种蛋白质 大亚基50S 1590kD 23S rRNA+5S rRNA 34种蛋白质 18S rRNA 小亚基40S kD 80S 4420kD 大亚基60S kD 33种蛋白质 28S rRNA+5.8S rRNA+5rRNS 45种蛋白质 • Redundancy of the genes coding for tRNA • Bacterial genome: 3 copies –primary transcript-30sRNA—enzymatically cleaved into three components---23S/16S/5S • Eukayotes: rRNA gene called rDNA, many copies, 20/drosophila 500/ X. laevis tandem repeats separated by spacer DNA 45S---28s/18/4.5s In human ,these gene clusters have been localized at near end of 13.14.15.21.22 chromosomes 5s rDNA tRNA structure • • • • • • • tRNA占17-18%,73-90nt, 1963, Holley first tRNA structure,250species。 tRNA: single strand 75-90tMW25000、4S unusual, rare, or odd, 7-15 modified bases 5´ phosophatation ,pG, 3 ´CCA-OH。 tRNA half of bases helix 4arms and 3loops, cloverleaf model。 1-甲基次黄(嘌呤核)苷酸 次黄(嘌呤核)苷酸 NN-二甲基鸟苷酸 1-甲基鸟苷酸 假尿嘧啶核酸 -Gp pCpCpA- tRNA functional sites anticodon aa attachment site additional arms assist binding to ribosome 3-D structure L-shape folded cloverleaf... but all unique recognized by specific aa synthetases unique anticodons tRNA genes Charging tRNA (Biochemistry) • Before translation can proceed, the tRNA molecules must be chemically linked to their respective amino acids. This activation process, called charging occurs under the direction of enzymes called aminoacyl tRNA synthetases . summary of chemical reactions synthetase1 1. aa1 + tRNA1 aa1tRNA1 peptidyl transferase (on ribosome) 2. aa1tRNA1 + aa2tRNA2 aa1aa2tRNA2 + tRNA1 peptidyl transferase (on ribosome) 3. aa1aa2tRNA2 + aa3tRNA3 aa1aa2aa3tRNA3 + tRNA2 ... summary of chemical reactions synthetase1 1. aa1 + tRNA1 aa1tRNA1 peptidyl transferase (on ribosome) 2. aa1tRNA1 + aa2tRNA2 aa1aa2tRNA2 + tRNA1 peptidyl transferase (on ribosome) 3. aa1aa2tRNA2 + aa3tRNA3 polypepti aa1aa2aa3tRNA3 + tRNA2 ... de 13.2 Translation process— Protein synthesis • • • • Initiation Elongaton Termination polyribosomes The small ribosomal subunit binds to several initiation factors, and this complex in turn binds to mRNA (step 1). In bacteria, this binding involves a sequence of up to six ribonucleotides (AGGAGG, not shown), which precedes the initial AUG start codon of mRNA. This sequence (containing only purines and called the Shine-Dalgarno sequence) base-pairs with a region of the 16S rRNA of the small ribosomal subunit, facilitating initiation. The increase of the growing polypeptide chain by one amino acid is called elongation. The sequence of the second triplet in mRNA dictates which charged tRNA molecule will become positioned at the A site (step 1). Once it is present, peptidyl transferase catalyzes the formation of the pep-tide bond, which links the two amino acids (step 2). This enzyme is part of the large subunit of the ribosome. At the same time, the covalent bond between the amino acid and the tRNA occupying the P site is hydrolyzed (broken). The product of this reaction is a dipeptide, which is attached to the 3;-end of tRNA still residing in the A site. UAG, UAA, or UGA. These codons do not specify an amino acid, nor do they call for a tRNA in the A site. These codons are called stop codons, termination codons, or nonsense codons. The finished polypeptide is therefore still attached to the terminal tRNA at the P site, and the A site is empty. The termination codon signals the action of GTP-dependent release factors, which cleave the polypeptide chain from the terminal tRNA, releasing it from the translation complex (step I). PROTEIN SYNTHESIS polypeptide synthesis on ribosomes (prokaryote e.g.) PROTEIN SYNTHESIS mRNA binds to 30S subunit PROTEIN SYNTHESIS tRNAs bind at 2 sites on the ribosome PROTEIN SYNTHESIS A site = entry site for arriving aatRNAs PROTEIN SYNTHESIS P site = position of growing aaaatRNA chain PROTEIN SYNTHESIS peptidyl transferase-catalysis of peptide bonds PROTEIN SYNTHESIS deacylated tRNA leaves P-site PROTEIN SYNTHESIS ribosome moves 1 codon in 3' direction on mRNA PROTEIN SYNTHESIS A-site left vacant for arriving aatRNAs PROTEIN SYNTHESIS 3 distinct stages of translation... protein synthesis 1. initiation 2. elongation 3. termination PROTEIN SYNTHESIS initiation initiation factors IF1, IF2, IF3 1st aa = modified MET... N-formylmethionine (fMET) PROTEIN SYNTHESIS initiation initiation factors IF1, IF2, IF3 1st aa = modified MET... N-formylmethionine (fMET) inserted by initiator tRNA... tRNAfMET recognizes start codon... AUG fMET start only... MET anywhere else PROTEIN SYNTHESIS initiation 30S + IF3... + mRNA = initiation complex IF2 + GTP + fMETtRNAfMET ... P site on mRNA IF2 & GTP split, ribosome assembled, IF2 & IF3 released PROTEIN SYNTHESIS initiation MET or fMET anticodons AUG (GUG) codons ? start codon preceded by Shine-Delgarno sequence... PROTEIN SYNTHESIS initiation MET or fMET anticodons AUG (GUG) codons ? start codon preceded by Shine-Delgarno sequence... pairs with 16S rRNA PROTEIN SYNTHESIS elongation elongation factors EF-Tu, EF-Ts, EF-G PROTEIN SYNTHESIS elongation EF-Tu + GTP + aatRNA ... A site EF-Ts mediates release/recycle of EF-Tu PROTEIN SYNTHESIS elongation aaaa aatRNA on A site by peptidyl transferase EF-G + GTP mediate ribosome translocation... PROTEIN SYNTHESIS elongation EF-G + GTP mediate ribosome translocation 5' 3' tRNA released from P site aaaatRNA on A site P site PROTEIN SYNTHESIS termination release factors RF1, RF2, RF3 RF1 & 2 recognize A site STOP RF1 ... UAA & UAG RF2 ... UAA & UGA aaaa released from P site ribosome dissociates Process Initiation Elongation Factor Role IF1 IF2 Stabilizes 305 subunit Binds fmet-tRNA to 30S-mRNA complex; binds to GTF and stimulate IF3 Binds 305 subunit to mRNA EF-Tu EF-Ts Binds GTP; Brings aminoacyl-tRNA to the A site of ribosome Generates active EF-Tu Stimulates translocation; GTP-dependent EF-G Terminate RFl release RF2 RF3 Catalyzes release of the polypeptide chain from tRNA an disaociation complex;like Behaves specific RPl; for Specific UAA for andUGA UAGand termination UAA codons codons Stimulates RF1 and RF2 PROTEIN SYNTHESIS how do mutations codon function ? ... sense (or functional) aa missense aa nonsense STOP... no aa PROTEIN SYNTHESIS Brenner, nonsense suppressor mutations in T4 phage normal STOP sequence PROTEIN SYNTHESIS Brenner, nonsense suppressor mutations in T4 phage mutate anticodon sequence in tRNATyr Polyribosome As elongation proceeds and the initial portion of mRNA has passed through the ribosome, this mRNA is free to associate with another small subunit to form a second initiation complex. This process can be repeated several times with a single mRNA and results in what are called polyribosomes or just polysomes. 13.3 Translation in Eukaryotes • The general features of the model we just discussed were initially derived from investigations of the translation process in bacteria. As we saw, one main difference between translation in prokaryotes and eukaryotes is that in the latter, translation occurs on ribosomes that are larger and whose rRNA and protein components are more complex than those of prokaryotes (see Figure 13-1). • Several other differences are also important. Eukaryotic mRNAs are much longer-lived than their prokaryotic counterparts. Most exist for hours rather than minutes prior to their degradation by nucleases in the cell, remaining available much longer to orchestrate protein synthesis. • Several aspects that involve the initiation of translation are different in eukaryotes. • First, the 5'-end is "capped" with a 7methylguanosine residue. The presence of this "cap" absent in prokaryotes. is essential to efficient translation, as RNAs lacking the cap are translated poorly. • In addition, most eukaryotic mRNAs contain a short recognition sequence that surrounds the initiating AUG codon—5'-ACCAUGG Marilyn Kozak, to function during initiation in the same way that the Shine-Dalgarno sequence functions in prokaryotic mRNA. Both greatly facilitate the initial binding of mRNA to the small subunit of the ribosome. • Another difference is that the amino acid formylmetbionine is not required to initiate eukaryotic translation. However, as in prokaryotes, the AUG triplet, which encodes methionine, is essential to the formation of the translational complex, and a unique transfer RNA (tRNAmet ) is used during initiation. 13.4 Proteins, Heredity, and Metabolism • Now let's consider how we know that proteins are the end products of genetic expression. The first insight into the role of proteins in genetic processes was provided by observations made by Sir Archibald Garrod and William Bateson early in the twentieth century. Garrod was born into an English family of medical scientists. His father was a physician with a strong interest in the chemical basis of rheumatoid arthritis, and his eldest brother was a leading zoologist in London. It is not surprising, then, that as a practicing physician, Garrod became interested in several human disorders that seemed to be inherited. 那么基因又是如何实现其功能的?在20 世纪初,英国医生A.Garrod首先发现人 类中几种先天性代谢缺陷疾病,如苯丙 酮尿症(phenylketonuria简称PKU)就 是由一个常染色体隐性基因决定的。 • Although he also studied albinism and cystinuria, we shall describe his alkaptonuria individuals afflicted with this disorder cannot metabolize the aikapton 2,5-dihydroxyphenylacetic acid, also known as homogentisic acid. As a result, an important metabolic pathway (Figure 13-10.) is blocked. Homogentisic acid accumulates in cells and tissues and is excreted in the urine. The molecule's oxidation products are black and easily detectable in the diapers of newborns. The products tend to accumulate in cartilaginous areas, causing a darkening of the ears and nose. In joints, this deposition leads to a benign arthritic condition. This rare disease is not serious, but it persists throughout an individual's life. 苯丙氨酸羧化酶 酪氨酸酶 人的先天代谢缺陷 • 1、苯丙氨酸的代谢 蛋白质 蛋白质 苯丙氨酸羧化酶 苯丙氨酸 苯丙酮酸 PKU PP 苯 丙 尿 症 酮 3,4二羟基 苯丙氨酸 酪氨酸 酪氨酸酶 对羟苯丙酮酸 尿黑酸 尿黑酸氧化酶 AKU Aa黑尿症 乙酰酯酸 CO2+H2O cc 白 化 病 黑色素 • 这个代谢途径任何地方出现问题则可能表现: • (1)黑尿症:病人没有不健康的地方,只是尿液在空气中 变黑。(正常人不会变黑,因为血液中有一种尿黑酸氧化 酶 • (2)白化病:患者不能形成黑色素 • (3)苯丙酮尿症:患者不能形成苯丙氨酸羧化酶——酪氨 酸。由于苯丙氨酸的累积可引起下列变化: • 过量苯丙氨酸损害中枢神经系统,影响智力发育 • 只能通过苯丙氨酸转氨酶作用——苯丙酮酸,小便排除 • 抑制酪氨酸形成,不能产生黑色素,患者肤色和发色浅。、 • 苯丙氨酸,蛋白质原料,人体必须氨基酸。因此婴儿期及 早诊断明确,可通过食物控制苯丙氨酸的摄入量,防止中 枢神经损害。 • 2、半乳糖代谢 • 不仅氨基酸,脂肪、糖类代谢也可能由于缺乏适当的酶而 受影响。 13.5 The One-Gene:One-Enzyme Hypothesis • In two separate investigations beginning in 1933, George Beadle was to provide the first convincing experimental evidence that genes are directly responsible for the synthesis of enzymes. The first investigation, conducted in collaboration with Boris Ephrussi, involved Drosophila eye pigments. Together, they confirmed that mutant genes that alter the eye color of fruit flies can be linked to biochemical errors that, in all likelihood, involve the loss of enzyme function. • Encouraged by these findings, Beadle then joined with Edward Tatum to investigate nutritional mutations in the pink bread mold Neurospora crassa. This one to one Beadle and Tatum: Neurospora Mutants • In the early 1940s. Beadle and Tatum chose to work with Neurospora. By inducing mutations, they produced strains that had genetic blocks of reactions essential to the growth of the organism. • Beadle and Tatum knew that this mold could manufacture nearly everything necessary for normal development. • This organism can synthesize 9 water-soluble vitamins, 20 amino acids, numerous carotenoid pigments, and all essential purines, and pyrimidines. Beadle and Tatum irradiated asexual conidia (spores) with X-rays to increase the frequency of mutations and allowed them to be grown on "complete" medium containing all the necessary growth factors (e.g., vitamins and amino acids, etc.). • Under such growth conditions,to grow by virtue of supplements present in the enriched complete medium. All the cultures were then transferred to minimal medium. • If growth on the minimal medium, they able to synthesize all the necessary growth factors themselves, and the researchers concluded that the culture did not contain a mutation. • If no growth occurred, then they concluded that the culture contained a nutritional mutation, and the only task remaining was to determine its type. Both cases are shown in Figure 13-1 l(a). • Many thousands of individual spores derived by this procedure were isolated and grown on complete medium. To identify the mutant type, the mutant strains were tested on a series of different minimal media [Figure 13-1 l(b) and (c)], each containing groups of supplements • Genes and Enzymes: Analysis of Biochemical Pathways The one-gene:one-enzyme concept and its attendant methods have been used over the years to work out many details of metabolism in Neurospora, Escherichia coli, and a number of other microorganisms. • One of the first metabolic pathways to be investigated in detail was that leading to the synthesis of the amino acid arginine in Neurospora. By studying seven mutant strains, each requiring arginine for growth (arg-). • Adrian Srb and Norman Horowitz ascertained a partial biochemical pathway that leads to the synthesis of this molecule. Their work demonstrates how genetic analysis can be used to establish biochemical information. • Srb and Horowitz tested each mutant strain's ability to reestablish growth if either citrulline or ornithine, two compounds with close chemical similarity to arginine, was used as a supplement to minimal medium. If either was able to substitute for arginine, they reasoned that it must be involved in the biosynthetic pathway of arginine. They found that both molecules could be substituted in one or more strains. Of the seven mutant strains, four of them (arg 4-7) grew if supplied with either citrulline, ornithine, or arginine. Two of them (arg 2 and arg 3) grew if supplied with citrulline or arginine. One strain (arg 1) would grow only if arginine were supplied; neither citrulline nor ornithine could substitute for it. From these experimental observations, the following pathway and metabolic blocks for each mutation were deduced: 生化突变与一基因一酶说 • • • • • • • 菌株 鸟氨酸 1 — 2-3 — 4-7 生长 基因: arg1 酶 : E1 前体 前体 鸟氨酸 瓜氨酸 精氨酸 — 生长 生长 生长 生长 生长 arg2 arg3 E2 E3 瓜氨酸 精氨酸。 Insights and Solutions 13.6 One-Gene:One-Polypeptide Chain • Two factors soon modified the one-gene:one-enzyme hypothesis. • First, while nearly all enzymes are proteins, not all proteins are enzymes. the more accurate phraseology one-gene :one-protein. • Second, proteins often show a subunit structure consisting of two or more polypeptide chains. Because each distinct polypeptide chain is encoded by a separate gene, a more modern statement of Beadle and Tatum's basic hypothesis is one-gene: one-polypeptide chain. • These modifications of the original hypothesis became apparent during the analysis of hemoglobin structure in individuals afflicted with sickle-cell anemia. • HbA由4条多肽链组成(α2β2), 2α链,各141AA 2β链, 146AA • Vernon M.Ingram证明HhA和HbS有相同的α链, 只是β链上第6位AA HbA是Glutamine,而HbS是Val。 • 因此HbA和HbS这对等位基因的差别导致了由该基因 所控制的多肽链上的一个氨基酸的差别。 由此可见基因是以某种方式规定了蛋白质中氨基酸顺 序的。 • 已发现由于α链或β链上不同氨基酸的变异导致许 多不同类型的血红蛋白。 • 如HbC也是由于β链上第6位Glu——Lys,纯合的 HbCHbC个体只表现轻度贫血。 • 还有HbHope异常的血红蛋白是由于β链第136位上 氨基酸发生变异,但不产生明显的临床症状。 13.11 Protein Domains and Exon Shuffling • We conclude our discussion of proteins by briefly discussing the important finding that regions made up of specific ammo acid sequences are associated with specific functions in protein molecules. Such sequences, usually between 50 and 300 amino acids, constitute what are called protein domains and are represented by modular portions of the protein that fold into stable, unique conformations independently of the rest of the molecule. Different domains impart different functional capabilities. Some proteins contain only a single domain, while others contain two or more. • The significance of domains rests at the tertiary structure level of proteins. Each such modular unit can be a mixture of secondary structures, including both a helicies and p pleated sheets. The unique conformation that is assumed in a single domain imparts a specific function to the protein. For example, a domain may serve as the catalytic basis of an enzyme, or it may impart the capability to bind to a specific ligand as part of a membrane or another molecule. Thus, in the study of proteins, you will hear of catalytic domains, DNA-binding domains, and so on. The result is that a protein must be looked at as being composed of a series of structural and functional modules. Obviously, the presence of multiple domains in a single protein increases the versatility of each molecule and adds to its functional • An interesting proposal to explain the genetic origin of protein domains was put forward by Waller Gilbert in 1977. Gilbert suggested that the functional regions of genes in higher organisms consist of collections of exons originally present in ancestral genes that are brought together through recombination during the course of evolution. Referring to this process as exon shuffling, Gilbert proposed that exons, like protein domains, are also modular. Gilbert proposed that during evolution, exons may have been reshuffled between genes in eukaryotes such that different genes share similar domains. • Since 1977, a serious research effort has been directed toward the analysis of gene structure. In 1985, more direct evidence in favor of Gilbert's proposal of exon modules was presented. For example, the human gene encoding the membrane receptor for low-density lipoproteins (LDL) was isolated and sequenced. The LDL receptor protein is essential to the transport of plasma cholesterol into the cell. It mediates endocytosis and is expected to have numerous functional domains. These include the capability of this protein to bind specifically to the LDL substrates and to interact with other proteins at different levels of the membrane during transport across it. hi addition, this receptor molecule is modified post-translationally by the addition of a carbohydrate; a domain must exist that links to this carbohydrate. Detailed analysis of the gene encoding this protein supports the concept of exon modules and their shuffling during evolution, The gene is quite large—45,000 nucleotides—and contains 18 exons. These represent only slightly less than 2600 nucleotides. These exons are related to the functional domains of the protein and appear to have been recruited from other genes during evolution. Figure 13-21 shows these relationships. • The first exon encodes a signal sequence that is removed from the protein before the LDL receptor becomes part of the membrane. The next five exons represent the domain specifying the binding site for cholesterol. This domain is made up of a sequence of 40 amino acids repeated seven times. The next domain consists of a sequence of 400 amino acids bearing a striking homology to the mouse peptide hormone epidermal growth factor (EOF). This region is encoded by eight exons and contains three repetitive sequences of 40 amino acids. A similar sequence is also found in three blood-clotting proteins. The fifteenth exon specifies the domain for the posttranslational addition of the carbohydrate, while the remaining two specify regions of the protein that are part of the membrane, anchoring the receptor to specific sites called coated pits on the cell surface.