分子生物學(xué)課件分子生物translation4

1、Termination of translationPart 4Three codons terminate protein synthesisUAAUGAUAGTermination codons are recognized by protein factorsTwo stages are involved in ending translation. The termination reaction itself involves release of the protein chain from the last tRNA. The post-termination reaction

2、involves release of the tRNA and mRNA, and dissociation of the ribosome into its subunitsclass 1 release factors (RF) recognize Termination codons and trigger hydrolysis of the peptide chain from the tRNA in the P site.RF1 recognizes UAA and UAG; RF2 recognizes UGA and UAA.The factors act at the rib

3、osomal A site and require polypeptidyl-tRNA in the P site.The class 1 release factors are assisted by class 2 release factors.class 2 release factors are not codon-specific. they are GTP-binding proteins. In E. coli, the role of the class 2 factor RF3 is to release the class 1 factor from the riboso

4、me after release of the polypeptide chain.eRF1The eukaryotic class 1 release factor, eRF1, is a single protein that recognizes all three termination codons. Its sequence is unrelated to the bacterial factors. It can terminate protein synthesis in vitro without the class 2 factor, eRF3, although eRF3

5、 is essential in yeast in vivo.How do release factors recognize stop codons?just three amino acids are responsible for the specificity of stop codon recognition. The region defined by these three amino acids represents a peptide anticodon that interacts with and recognizes stop codons.All class I fa

6、ctors share a conserved three-amino acid sequence (glycine glycine glutamine, GGQ) that is essential for polypeptide release.the GGQ motif is located in close proximity to the peptidyl transferase centerthe glutamine (Q) to position a water molecule to substitute for the amino acid of aminoacyl-tRNA

7、 in the peptidyl transfer reaction.GDP/GTP Exchange and GTP Hydrolysis Controlthe Function of the Class II Release FactorRF3 is a GTP-binding protein but has a higher affinity for GDP than GTP. Thus, free RF3 is predominantly in the GDP-bound form. RF3-GDP binds to the ribosome in a manner that depe

8、nds on the presence of a class I release factor. After the class I RF stimulates polypeptide release, a change in the conformation of the ribosome and the class 1 factor stimulates RF3 to exchange its bound GDP for a GTP. The binding of GTP to RF3 leads to the formation of a high-affinity interactio

9、n with the ribosome that displaces the class I factor from the ribosome. This change also allows RF-3 to associate with the factor binding center of the large subunit.西北農(nóng)林科技大學(xué) 郭澤坤In the absence of a bound class I factor,RF3-GDP has a low affinity for the ribosome and is released.ribosome recycling.I

10、n prokaryotic cells a factor known as the ribosome recycling factor (RRF) cooperates with EF-G and IF3 to recycle ribosomes after polypeptide releaseRRF has a structure that mimics tRNA, except that it lacks an equivalent for the 3 amino acid-binding regionRRF is closely associated only with the lar

11、ge subunit portion of the A site,RRF acts on the 50S subunit, and IF-3 acts on the 30S subunit to remove deacylated tRNA RF3 resembles the GTP-binding domains of EF-Tu and EF-G,RF1/2 resemble the C-terminal domain of EF-G, which mimics tRNA.these factors all have the same general shape and bind to t

12、he ribosome successively at the same site (basically the A site or a region extensively overlapping with it)Termination Codons Are Recognized by Protein FactorsFunctional homologies of prokaryotic and eukaryotic translation factors.TRANSLATION-DEPENDENT REGULATIONOF mRNA AND PROTEIN STABILITYAt some

13、 frequency, mRNAs will be made that are mutant or damaged. Such defective mRNAs can arise from mistakes in transcription or from damage that occurs after they are synthesized. Such damaged mRNAs have the possibility of making plete or incorrect proteins that could have negative effects on the cell.

14、In some cases, such as point mutations that change only a single amino acid, there is little that can be done to eliminate the mutant mRNA or its protein product.The SsrA RNA Rescues Ribosomes that Translate Broken mRNAsWhat happens to a ribosome that initiates translation of an mRNA fragment that l

15、acks a termination codon in the appropriate reading frame?In prokaryotic cells, such stalled ribosomes are rescued by the action of a chimeric RNA molecule that is part tRNA and part mRNA. called a tmRNA.SsrA is a 457-nucleotide tmRNA that includes a region at its 3 end that strongly resembles tRNAA

16、la。This similarity allows the SsrA RNA to be charged with alanine and to bind EF-Tu-GTPWhen a ribosome is stalled at the 3 end of an mRNA, the SsrAAla-EF-TU-GTP complex binds to the A site of the ribosome and participates in the peptidyl transferase reactionTranslocation of the peptidy-SsrA RNA resu

17、lts in the release of the broken mRNA.Interestingly, the ten-amino-acid tag is recognized by cellular proteases that rapidly degrade the tag and the truncated polypeptide to which it is attached.to prevent these defective proteins from harming the cell.蛋白質(zhì)前體的加工新生多肽鏈大多數(shù)是沒有功能的，必須經(jīng)過加工修飾才能轉(zhuǎn)變?yōu)橛谢钚缘牡鞍踪|(zhì)。 N端

18、fMet或Met的切除二硫鍵的形成特定氨基酸的修飾。氨基酸側(cè)鏈的修飾包括：磷酸化（如核糖體蛋白質(zhì)）、糖基化（如各種糖蛋白） 、甲基化（如組蛋白、肌肉蛋白質(zhì)） 、乙基化（如組蛋白） 、羥基化（如膠原蛋白）和羧基化等。切除新生鏈中非功能片段1 N端fMet或Met的切除 不管是原核生物還是真核生物，N端的甲硫氨酸往往在多肽鏈合成完畢之前就被切除。二硫鍵的形成又稱SS鍵。是2個(gè)SH基被氧化而形成的SS形式的硫原子間的鍵。在生物化學(xué)的領(lǐng)域中，通常系指在肽和蛋白質(zhì)分子中的半胱氨酸殘基中的鍵。此鍵在蛋白質(zhì)分子的立體結(jié)構(gòu)形成上起著一定的重要作用。為了確定蛋白質(zhì)的一級(jí)結(jié)構(gòu)，首先必須將二硫鍵打開，使成為線狀多肽

19、鏈。 特定氨基酸的修飾磷酸化（如核糖體蛋白質(zhì)）;糖基化（如各種糖蛋白）;甲基化（如組蛋白、肌肉蛋白質(zhì)）;乙基化（如組蛋白）;羥基化（如膠原蛋白）;羧基化4 切除新生鏈中非功能片段前胰島素原的加工蛋白質(zhì)的折疊新生多肽鏈必須經(jīng)過正確的折疊，才能形成動(dòng)力學(xué)和熱力學(xué)穩(wěn)定的三維構(gòu)象，從而表現(xiàn)出生物學(xué)活性或功能。多肽鏈的折疊式一個(gè)復(fù)雜的過程，新生肽鏈一般首先折疊成二級(jí)結(jié)構(gòu)，再進(jìn)一步折疊盤繞成三級(jí)結(jié)構(gòu)。對(duì)于寡聚蛋白質(zhì)，一般還需要組裝成更為復(fù)雜的四級(jí)結(jié)構(gòu)。分子伴侶是能在細(xì)胞內(nèi)輔助新生肽鏈正確折疊的一類蛋白質(zhì)。它是一類在序列上沒有相關(guān)性但有共同功能的保守性蛋白質(zhì)。分為兩類：熱休克蛋白、伴侶素。蛋白質(zhì)轉(zhuǎn)運(yùn)機(jī)制翻譯

20、-轉(zhuǎn)運(yùn)同步機(jī)制 蛋白質(zhì)的合成和運(yùn)轉(zhuǎn)同時(shí)發(fā)生。分泌蛋白質(zhì)大多是以同步機(jī)制運(yùn)輸?shù)摹７g后轉(zhuǎn)運(yùn)機(jī)制 蛋白質(zhì)從核糖體上釋放后才發(fā)生運(yùn)轉(zhuǎn)。在細(xì)胞器發(fā)育過程中，由細(xì)胞質(zhì)進(jìn)入細(xì)胞器的蛋白質(zhì)大多是以翻譯后運(yùn)轉(zhuǎn)機(jī)制運(yùn)輸?shù)摹７g-轉(zhuǎn)運(yùn)同步機(jī)制機(jī)制過程：分泌蛋白的生物合成開始于核糖體，翻譯到大約50個(gè)氨基酸殘基，信號(hào)肽開始從核糖體的大亞基露出，被內(nèi)質(zhì)網(wǎng)膜上的受體識(shí)別并與之結(jié)合。信號(hào)肽過膜后被內(nèi)質(zhì)網(wǎng)腔的信號(hào)肽酶水解，新生肽隨之通過蛋白孔道穿越疏水的雙層磷脂。當(dāng)核糖體移到mRNA的“終止”密碼子，蛋白質(zhì)合成即告完成，翻譯體系解散，膜上的蛋白孔道消失，核糖體重新處于自由狀態(tài)。 蛋白質(zhì)定位的信息存在于該蛋白質(zhì)自身結(jié)構(gòu)中，并

21、且通過與膜上特殊受體的相互作用得以表達(dá)。信號(hào)肽緊接在起始密碼之后，大部分是疏水氨基酸。信號(hào)肽的長度在1336個(gè)殘基。 信號(hào)肽的特點(diǎn)：1015個(gè)疏水氨基酸；N端有帶正電荷的氨基酸；C端接近切割點(diǎn)處帶數(shù)個(gè)極性氨基酸；離切割點(diǎn)最近的氨基酸往往帶有很短的側(cè)鏈（Ala, Gly）。蛋白合成-定位內(nèi)質(zhì)網(wǎng)1. 完整的信號(hào)肽是保證蛋白質(zhì)轉(zhuǎn)運(yùn)的必要條件。SRP：信號(hào)識(shí)別蛋白2. 僅有信號(hào)肽還不足以保證蛋白質(zhì)轉(zhuǎn)運(yùn)的發(fā)生。3. 信號(hào)序列的切除并不是轉(zhuǎn)運(yùn)所必需的。4. 并非所有的轉(zhuǎn)運(yùn)蛋白都有可降解的信號(hào)肽。新生蛋白質(zhì)通過同步轉(zhuǎn)運(yùn)途徑進(jìn)入內(nèi)質(zhì)網(wǎng)內(nèi)腔的主要過程翻譯后轉(zhuǎn)運(yùn)機(jī)制葉綠體和線粒體中有許多蛋白質(zhì)和酶是由細(xì)胞質(zhì)提供的

22、，其中絕大多數(shù)以翻譯后運(yùn)轉(zhuǎn)機(jī)制進(jìn)入細(xì)胞器內(nèi)。線粒體蛋白質(zhì)跨膜轉(zhuǎn)運(yùn)特征:通過線粒體膜的蛋白質(zhì)在運(yùn)轉(zhuǎn)之前大多數(shù)以前體形式存在，它由成熟蛋白質(zhì)和位于N端的一段前導(dǎo)肽（leader peptide）共同組成，前導(dǎo)肽約含2080個(gè)氨基酸殘基，當(dāng)前體蛋白過膜時(shí)，前導(dǎo)肽被多肽酶所水解，釋放成熟蛋白質(zhì)。蛋白質(zhì)通過線粒體膜運(yùn)轉(zhuǎn)是一種需要能量的過程。蛋白質(zhì)通過線粒體膜運(yùn)轉(zhuǎn)時(shí)，首先由外膜上的Tom受體復(fù)合蛋白識(shí)別與分子伴侶相結(jié)合的待運(yùn)轉(zhuǎn)多肽，通過Tom和Tim組成的膜通道進(jìn)入線粒體內(nèi)腔。蛋白質(zhì)跨膜運(yùn)轉(zhuǎn)時(shí)的能量來自線粒體Hsp70引發(fā)的ATP水解和膜電位差。 前導(dǎo)肽的作用與性質(zhì)擁有前導(dǎo)肽的線粒體蛋白質(zhì)前體能夠跨膜運(yùn)轉(zhuǎn)

23、進(jìn)入線粒體，在這一過程中前導(dǎo)肽被水解，前體轉(zhuǎn)變?yōu)槌墒斓鞍?，則失去繼續(xù)跨膜能力。因此，前導(dǎo)肽對(duì)線粒體蛋白質(zhì)的識(shí)別和跨膜運(yùn)轉(zhuǎn)起著關(guān)鍵作用。 前導(dǎo)肽具有如下特性：帶正電荷的堿性氨基酸（特別是精氨酸）含量較為豐富，它們分散于不帶電荷的氨基酸序列之間；缺少帶負(fù)電荷的酸性氨基酸；羥基氨基酸（特別是絲氨酸）含量較高；有形成兩親（既有親水又有疏水部分）-螺旋結(jié)構(gòu)的能力。葉綠體蛋白質(zhì)的跨膜轉(zhuǎn)運(yùn)葉綠體多肽在胞質(zhì)中的游離核糖體上合成后脫離核糖體并折疊成具有三級(jí)結(jié)構(gòu)的蛋白質(zhì)分子，多肽上某些特定位點(diǎn)結(jié)合于只有葉綠體膜上才有的特異受體位點(diǎn)。葉綠體定位信號(hào)肽一般有兩個(gè)部分，第一部分決定該蛋白質(zhì)能否進(jìn)入葉綠體基質(zhì)，第二部分決

24、定該蛋白能否進(jìn)入類囊體。葉綠體蛋白質(zhì)轉(zhuǎn)運(yùn)有如下特點(diǎn)：活性蛋白水解酶位于葉綠體基質(zhì)內(nèi)。葉綠體膜能夠特異地與葉綠體蛋白的前體結(jié)合。葉綠體前體內(nèi)可降解序列因植物和蛋白質(zhì)種類不同而表現(xiàn)出明顯的差異。核定位蛋白的轉(zhuǎn)運(yùn)機(jī)制為了核蛋白的重復(fù)定位，這些蛋白質(zhì)中的信號(hào)肽 被稱為核定位序列（NLSNuclear Localization Sequence）一般都不被切除。NLS可以位于核蛋白的任何部位。蛋白質(zhì)向核內(nèi)運(yùn)輸過程需要核運(yùn)轉(zhuǎn)因子（Importin）、和一個(gè)低分子量GTP酶（Ran）參與。核定位蛋白跨細(xì)胞核膜運(yùn)轉(zhuǎn)過程示意圖蛋白質(zhì)的降解大腸桿菌蛋白的降解需要一個(gè)依賴于ATP的蛋白酶（稱為Lon）。真核生物蛋白

25、降解依賴于泛蛋白（ubiquitin）。蛋白質(zhì)的半衰期大約從30s到許多天不等。像血紅蛋白這樣少數(shù)蛋白的半衰期與細(xì)胞周期同樣長。成熟蛋白N端的第一個(gè)氨基酸（除已被切除的N端甲硫氨酸之外，但包括翻譯后修飾產(chǎn)物）在蛋白的降解中的影響很大。當(dāng)某個(gè)蛋白質(zhì)的N端是甲硫氨酸、甘氨酸、丙氨酸、絲氨酸、蘇氨酸和纈氨酸時(shí)，表現(xiàn)穩(wěn)定。其N端為賴氨酸、精氨酸時(shí)，表現(xiàn)最不穩(wěn)定，平均2-3min就被降解了。 蛋白質(zhì)的半衰期與N-端氨基酸殘基的關(guān)系A(chǔ)BCDpCMVGFPMrp1 (1-200)Mrp1 (201-600)Mrp1 (901-1528)BGHPAABCDGFPDAPIMergedGFPctMTSGFPctM

26、TSGFPadrMTSGFPadrRelated Codons Represent Chemically Similar Amino Acids Sixty-one of the sixty-four possible triplets code for twenty amino acids.Three codons (stop codons) do not represent amino acids and cause termination.The genetic code is tripletRelated Codons Represent Chemically Similar Amin

27、o Acids The genetic code was frozen at an early stage of evolution and is universal.Most amino acids are represented by more than one codon.Amino acids have 1-6 codons eachRelated Codons Represent Chemically Similar Amino AcidsThe multiple codons for an amino acid are synonymous and usually related.

28、third-base degeneracy The lesser effect on codon meaning of the nucleotide present in the third (3) codon position.Chemically similar amino acids often have related codons, minimizing the effects of mutation. CodonAnticodon Recognition Involves Wobbling Multiple codons that represent the same amino

29、acid most often differ at the third base position (the wobble hypothesis).Third bases have the least influence on codon meaningsCodonAnticodon Recognition Involves Wobbling The wobble in pairing between the first base of the anticodon and the third base of the codon results from looser monitoring of

30、 the pairing by rRNA nucleotides in the ribosomal A site. Wobble in base pairing allows G-U pairs to form Codonanticodon pairing involves wobbling at the third positionNovel Amino Acids Can Be Inserted at Certain Stop Codons The insertion of selenocysteine at some UGA codons requires the action of a

31、n unusual tRNA in combination with several proteins.The unusual amino acid pyrrolysine can be inserted at certain UAG codons.The UGA codon specifies both selenocysteine and cysteine in the ciliate Euplotes crassus. SelB is specific for Seleno-Cys-tRNASuppressor tRNAs Have Mutated Anticodons That Rea

32、d New Codons A suppressor tRNA typically has a mutation in the anticodon that changes the codons to which it responds.Suppressor tRNAs Have Mutated Anticodons That Read New Codons When the new anticodon corresponds to a termination codon, an amino acid is inserted and the polypeptide chain is extend

33、ed beyond the termination codon.This results in nonsense suppression at a site of nonsense mutation, or in readthrough at a natural termination codon.Missense mutations change a single codon so as to cause the replacement of one amino acid by another in a protein sequence.A nonsense mutation is any

34、change in DNA that replaces a codon specifying an amino acid with a translation-termination codon (UAG, UGA, or UAA).In bacteria they are used most often with relative frequencies UAAUGAUAG.UGA is used more heavily than UAG, although there appear to be more errors reading UGA. (An error in reading a

35、 termination codon, when an aminoacyl-tRNA improperly responds to it, results in the continuation of protein synthesis until another termination codon is encountered.)Nonsense mutations can be suppressed by a tRNA with a mutant anticodonSuppressor tRNAs Have Mutated Anticodons That Read New CodonsMi

36、ssense suppression occurs when the tRNA recognizes a different codon from usual, so that one amino acid is substituted for another. Missense suppressors compete with wild typeThere Are Nonsense Suppressors for Each Termination Codon Each type of nonsense codon is suppressed by a tRNA with a mutated

37、anticodon.Some rare suppressor tRNAs have mutations in other parts of the molecule. Suppressors have anticodon mutationsSuppressors May Compete with Wild-Type Reading of the Code Suppressor tRNAs compete with wild-type tRNAs that have the same anticodon to read the corresponding codon(s).Efficient s

38、uppression is deleterious because it results in readthrough past normal termination codons.The UGA codon is leaky and is misread by Trp-tRNA at 1% to 3% frequency. Nonsense suppressors read through natural termination codonsThe Ribosome Influences the Accuracy of Translation The structure of the 16S rRNA at the P and A sites of the ribosome influences the accuracy of translation. The ribosome selects aminoacyl-tRNAsFrameshifting Occurs at Slippery Sequences The reading frame may be influenced by the sequence of mRNA and the ribosomal environment.