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Copyright © 2013 Pearson Canada Inc. 28 - 1 Chapter 28 Information Decoding: Translation and Post-translational Protein Processing 授課老師:劉浩屏 Office: 診中.

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Presentation on theme: "Copyright © 2013 Pearson Canada Inc. 28 - 1 Chapter 28 Information Decoding: Translation and Post-translational Protein Processing 授課老師:劉浩屏 Office: 診中."— Presentation transcript:

1 Copyright © 2013 Pearson Canada Inc Chapter 28 Information Decoding: Translation and Post-translational Protein Processing 授課老師:劉浩屏 Office: 診中 Tel: (04) Website:

2 Copyright © 2013 Pearson Canada Inc Outline: An Overview of Translation The Genetic Code The Major Participants in Translation: mRNA, tRNA, and Ribosomes Mechanism of Translation Inhibition of Translation by Antibiotics Translation in Eukaryotes Rates and Energetics of Translation The Final Stages in Protein Synthesis: Folding and Covalent Modification Protein Targeting in Eukaryotes The Fate of Proteins: Programmed Destruction

3 An overview of translation Copyright © 2013 Pearson Canada Inc Translation of an RNA message into a protein: As the ribosome moves along the message, it accepts specific aminoacyl tRNAs in succession, selecting them by matching the trinucleotide anticodon on the tRNA to the trinucleotide codon on the RNA message( step 1 ). The amino acid (the second one of the chain, Val) accepts the growing polypeptide chain (the previously bound fMet, step 2 ). The ribosome moves on to the next codon to repeat the process, while releasing the deacylated transfer RNA that held the growing peptide in the previous cycle (the tRNA for fMet, step 3 ). The preceding steps are repeated, adding more amino acids to the chain, until a stop signal is read ( step 4 ), where upon a protein release factor causes both the polypeptide and the mRNA to be released.

4 Transfer RNA (tRNA) Copyright © 2013 Pearson Canada Inc Transfer RNAs are the adaptor molecules that match amino acid to codon. Activation of amino acids for incorporation into proteins: A specific enzyme, aminoacyl-tRNA synthetase, recognizes both a particular amino acid and a tRNA carrying the corresponding anticodon. This synthetase catalyzes the formation of an aminoacyl tRNA, with accompanying hydrolysis of one ATP to AMP. Messenger RNA is read 5’  3’. Polypeptide synthesis begins at the N -terminus.

5 The genetic code Copyright © 2013 Pearson Canada Inc Three conceivable kinds of genetic codes: Early research on the nature of the code quickly showed that a non-overlapping, unpunctuated code (c) fit all experimental observations.

6 Copyright © 2013 Pearson Canada Inc Use of synthetic polynucleotides with repeating sequences to decipher the code: This example shows how polypeptides derived from the (AAG) n polymer were used to confirm the triplet code and help identify codons. The polymer (AAG) n can yield three different polypeptides, depending on which reading frame is employed. The genetic code

7 Copyright © 2013 Pearson Canada Inc The genetic code (as written in RNA): The genetic code as used in most organisms are shown. When AUG is used as a start codon, it codes for N-formylmethionine (fMet) in prokaryotes or methionine (Met) in eukaryotes. The code is redundant (e.g. Leu, Arg). Several codons may correspond to a single amino acid, sometimes via wobble in the 5’ anticodon position. UAA, UAG, and UGA are nonsense codons which do not code for any amino acid. Features of the genetic code

8 Copyright © 2013 Pearson Canada Inc Amino acid sequence analysis of T4 phage lysozyme mutants : e is the gene for lysozyme, a portion of whose amino acid sequence is shown. One of two proflavin-induced mutations, either eJ42 or eJ44 disrupted the reading frame by deleting one base pair, and a second mutation restored the wild-type reading frame by inserting a base pair, but altering the amino acid sequence between the two mutant sites. The mRNA sequences encoding these five altered amino acids are inferred from the genetic code and the known action of the mutagen. Validation of the genetic code wild-type mutant mutagen

9 Copyright © 2013 Pearson Canada Inc Modifications of the genetic code An adaption within mitochondria to oxidative stress 22 nd amino acid 21 st amino acid One codon codes for two amino acids

10 Copyright © 2013 Pearson Canada Inc The “21 st ”amino acids: selenocysteine (Sec) −The condon UGA has a Sec Insertion Sequence (SECIS). −There are 25 selenocysteine proteins. −Some of them participate in oxidant protection. The “22 nd ” amino acid: pyrrolysine (Pyl) −It has a much narrower distribution. Deviations from the genetic code

11 Copyright © 2013 Pearson Canada Inc In general, each amino acid is characterized by the first two codon letters. Redundancy is usually expressed in the third letter. A single tRNA may recognize several different codons. The recognition involves 3’ residue of the codon and 5’ residue of the anticodon. The wobble hypothesis: The 5’ base of the anticodon is capable of wobble in its position during translation, allowing it to make non-Watson-Crick hydrogen-bonding arrangement with several codon bases. The wobble hypothesis mRNA (3’) tRNA (5’) Watson-Crick hydrogen bonding Non-Watson-Crick hydrogen bonding

12 Copyright © 2013 Pearson Canada Inc The wobble pairs (inosine) (wobble position)

13 Copyright © 2013 Pearson Canada Inc In almost all organisms, UAA, UAG, and UGA are used for stop signals. As the message begins to be read, the first AUG encountered is interpreted as a start signal. All proteins start with N-fMet in prokaryotes or Met in eukaryotes, at least when they are first synthesized. In prokaryotes, AUU, GUG, or UUG serves as start codon when located near the 5’ end of a message and codes for N-fMet. Stopping and starting

14 The major participants in translation: mRNA, tRNA, and ribosomes Copyright © 2013 Pearson Canada Inc The lac operon mRNA: The mRNA for the E. coli lac operon is about 5300 nucleotides long and contains the open reading frames for the lacZ, lacY, and lacA genes, each flanked appropriately by start, stop, and Shine–Dalgarno (SD) sequences. (mRNA)

15 Copyright © 2013 Pearson Canada Inc The major participants in translation: mRNA, tRNA, and ribosomes A Shine–Dalgarno (SD) sequence can base-pair with a sequence contained in the ribosomal RNA (16S) to produce a proper alignment for starting translation.

16 Copyright © 2013 Pearson Canada Inc All tRNAs share a general common structure that includes an anticodon loop, which pair with codons, and an acceptor stem, to which the amino acid is attached. Secondary structure of tRNAs (cloverleaf models): a)Generalized tRNA structure. b)A leucine tRNA from E. coli. c)A human mitochondrial tRNA for lysine. The major participants in translation: mRNA, tRNA, and ribosomes The tRNA specific to a given amino acid is designed by writing the amino acid as a superscript, e.g. tRNA Leu.

17 Copyright © 2013 Pearson Canada Inc The tRNAs are unique among RNA molecules in their high content of unusual and modified bases. Biosynthesis of these modified bases always occurs post- transcriptionally. E.g. an isomerase converts a uridine residue (1-ribosyluracil) to the unusual C-glycoside pseudouridine (5-ribosyluracil). The major participants in translation: mRNA, tRNA, and ribosomes psai

18 Copyright © 2013 Pearson Canada Inc Unusual base pairings in tRNA: (a, b) Some unusual pair matches. (c, d) Some examples of triple interactions. R represents the ribosyl residue of the RNA chain. The bases prefixed by m are methylated at the carbon atom corresponding to the superscript. Numbers following the letters designating bases show the position in the sequence. The major participants in translation: mRNA, tRNA, and ribosomes

19 Copyright © 2013 Pearson Canada Inc In step 1 the amino acid, which is bound to the AARS, is activated by ATP to form an aminoacyl adenylate intermediate. In step 2 the proper tRNA is accepted by the AARS, and the amino acid residue is transferred from the intermediate to the 3’ OH of the 3’-terminal residue of the tRNA (to form the 3’-aminoacyl tRNA by class II AARS) or to the 2’ OH (to form the 2’-Aminoacyl tRNA by class I AARS). Formation of aminoacyl tRNAs by aminoacyl tRNA synthetase (AARS) isomerization ATP 3’ Amino acid The 2’-Aminoacyl tRNA is subsequently isomerized to the 3’-aminoacyl tRNA. AARSs contribute toward the fidelity of translation by a process akin to proofreading by DNA polymerases (error frequency for protein synthesis of ~10 -4 ).

20 Copyright © 2013 Pearson Canada Inc Major “identity elements” in some tRNAs: Red circles represent the positions that have been shown to identify the tRNA to its cognate synthetase. Shown also is a synthetic polynucleotide containing the G-U alanine identity element (in red), which is a good substrate for alanyl-tRNA synthetase. Identity elements in tRNA identify it to its cognate synthetase (AARS) synthetic identity elements

21 Copyright © 2013 Pearson Canada Inc The E. coli glutaminyl tRNA synthetase coupled with its tRNA and ATP : The tRNA is represented by a detailed atomic model, the protein by its solvent-accessible surface (blue). The ATP (green) and the 3’ acceptor stem of the tRNA fit into a deep cleft in the synthetase. This cleft will also accommodate the amino acid (glutamine). This is a monomeric class I synthetase. The structure of a class I AARS–tRNA complex ATP tRNA Glu synthetase 3’ acceptor stem cleft

22 Copyright © 2013 Pearson Canada Inc Yeast aspartyl tRNA synthetase complexed with two molecules of tRNA Asp : This is a dimeric class II synthetase. Only one of the two tRNA molecules is bound in a catalytically productive confirmation. A class II AARS complexed with two molecules of tRNA tRNA Asp synthetase

23 Components of ribosomes Copyright © 2013 Pearson Canada Inc The ribosome is a large ribonucleoprotein particle containing 60-70% RNA and 30-40% protein. Ribosomes and their subunits are characterized in terms of their sedimentation coefficients (S) in ultracentrifugation. Bacterial and eukaryotic ribosomes are assembled along the same structural plan, with eukaryotic ribosomes being somewhat larger and more complex. S1~S21S1~S33L1~L34L1~L49 Bacterialeukaryotic

24 Copyright © 2013 Pearson Canada Inc In vivo assembly map, as determined by Williamson and colleagues Parallel pathways begin, with proteins added at either the 5’ domain or the 3’ domain of 16S rRNA. Assembly map for the 30S subunit

25 Copyright © 2013 Pearson Canada Inc Secondary structure of E. coli 16S rRNA

26 Copyright © 2013 Pearson Canada Inc A model of the 70S ribosome based upon early structural data: This model shows all three tRNA-binding sites occupied simultaneously, which does not normally occur. ○ E, the exit ○ P, the peptidyl-tRNA binding site ○ A, the aminoacyl-tRNA binding site This view has the 30S subunit in front and the 50S subunit to the rear. A model of the 70S ribosome 30S subunit 50S subunit mRNA tRNA-binding sites exits through a tunnel in the 50S subunit creates peptide bonds, occurring at a site on the 50S

27 Copyright © 2013 Pearson Canada Inc A high-resolution model of the 50S ribosomal subunit: This view shows the two stalks and central protuberance (CP), seen also in the early electron micrographs. In this image RNA is in gray, and proteins are in gold. The peptidyltransferase site, in green, is identified from the binding of an inhibitor. This structural work establishes conclusively that the ribosome is a ribozyme. A high-resolution model of the 50S ribosome subunit The peptidyltransferase site By T.A. Steitz et al. (2009 Nobel Prize in Chemistry)

28 Copyright © 2013 Pearson Canada Inc The 30S subunit is in light blue—green (RNA) and blue (protein). The 50S subunit is in orange— brown (protein). Two bound tRNAs can be seen—peptidyl-tRNA in green and deacylated tRNA in yellow. mRNA is shown in gray. A model of the 70S ribosome, with mRNA and tRNA bound By V. Ramakrishnan et al. (2009 Nobel Prize in Chemistry)

29 Mechanism of translation Copyright © 2013 Pearson Canada Inc Translation involves three steps: o Initiation o Elongation o Termination Each aided by soluble protein factors (IFs, EFs, and RFs, respectively).

30 Copyright © 2013 Pearson Canada Inc The ribosomes and its associated factors There are some soluble factors that participate in the three stages of translation: i nitiation f actors ( IFs ), e longation f actors ( EFs ), and r elease f actors ( RFs ).

31 Initiation of prokaryotic translation Copyright © 2013 Pearson Canada Inc In initiation, the correct attachment of mRNA to the ribosome is determined by binding of the Shine– Dalgarno (SD) sequence to a sequence on the 16S rRNA of the ribosome.

32 Copyright © 2013 Pearson Canada Inc. Initiation results in formation of a 70S ribosome complex, which consists of a ribosome bound to mRNA and to a charged initiator tRNA (fMet-tRNA). IF1 and IF3 dissociate the 30S subunit from the preexisting 70S ribosome, thereby producing the free 30S subunits needed for initiation. IF2 (a G protein) delivers the fMet- tRNA fMet in binding to the 30S subunit, to which mRNA is bound at about the same time. The initiator AUG codon is positioned (by the presence upstream of a Shine- Dalgarno sequence) so that fMet-tRNA binds in the P site. The 30S initiation complex is formed completely, which has high affinity for a 50S subunit. Initiation of protein biosynthesis in prokaryotes Three tRNA binding sites: E, the exit P, the peptidyl-tRNA binding site A, the aminoacyl-tRNA binding site

33 The initiator tRNA in prokaryotes Copyright © 2013 Pearson Canada Inc The initiator tRNA is special since it recognizes and binds to the AUG codon that would normally code for methionine, but it actually carries an N -formylmethionine. The formyl group is added after charging of the tRNA, by an enzyme ( transformylase ) that recognizes the particular tRNA fMet and transfers a formyl group from 10- formyltetrahydrofolate.

34 Environment of tRNAs at the ribosome as determined by cross-linking Copyright © 2013 Pearson Canada Inc Cross-links from defined nucleotide positions in the tRNA to ribosomal proteins are shown. Proteins were differentially cross-linked depending on the location of the tRNA. The 30S subunit The 50S subunit Anticodon Acceptor stem

35 Copyright © 2013 Pearson Canada Inc Chain elongation in prokaryotic translation Growth of the polypeptide chain on the ribosome occurs by a cyclic processes. The first reaction in the overall elongation process would have been reaction between aa 2 -tRNA in the A site and fMet-tRNA (aa 1 -tRNA) in the P site. Following translocation ( step 3 ) and tRNA release ( step 4 ), the ribosome is ready to accept the next aminoacyl tRNA (aa- tRNA) and repeat the cycle. The cycles will continue until a termination codon is reached. This step is catalyzed by peptidyltransferase, an integral part of the 50S subunit ( the RNA portion ).

36 Regeneration of EF-Tu–GTP by EF–Ts exchange Copyright © 2013 Pearson Canada Inc This figure gives details of the regeneration cycle. Binding of the factor EF-Ts to EF-Tu allows the release of GDP and binding of a new GTP to prepare EF-Tu for another cycle. (step 1)

37 The ribosomal decoding pathway Copyright © 2013 Pearson Canada Inc a)L7/L12 stalk on 50S subunit recruits ternary complex ( aminoacyl-tRNA - EF-Tu - GTP ) to a ribosome with deacylated tRNA in E site and peptidyl- tRNA in P site. b)tRNA samples codon–anticodon pairing. A/T tRNA is a tRNA temperately distorted, to interact simultaneously with the decoding center of 30S subunit and EF-Tu. c)The match is sensed by specific nucleotides in coding site ( ① ). Codon recognition triggers 30S subunit domain closure. A chain of conformational changes ( ② - ⑤ ) allows EF-Tu to initiate GTP hydrolysis. (step 1) distort

38 The ribosomal decoding pathway Copyright © 2013 Pearson Canada Inc d)GTP hydrolysis and Pi release cause conformational change in EF-Tu, leading to its release from the ribosome. e)EF-Tu release leads to relaxation of aminoacyl-tRNA structure and its accommodation. f)Aminoacyl-tRNA accommodates at both the coding site and the peptidyltransferase site. (step 1)

39 Copyright © 2013 Pearson Canada Inc A mechanism for peptidyltransferase A conserved AMP residue on the RNA of the 50S subunit The ribosome is a ribozyme. (deacylated tRNA) (step 2) P site A site

40 Hybrid states in the translocation Copyright © 2013 Pearson Canada Inc. In elongation, the growing peptide chain at the P site is transferred to the newly arrived aminoacyl-tRNA in the A site. Translocation then moves this tRNA to the P site and the previous tRNA to the E site (hybrid state). EF-G -GTP moves temperately into the A site, facilitating the displacement of the peptidyl-tRNA complex. EF-G -GTP (step 3)

41 Copyright © 2013 Pearson Canada Inc The striking structural similarity between the EF-G and the ternary complex aa- tRNA-EF-Tu–GTP aa-tRNA - EF-Tu –GTP EF-G –GTP

42 EF-G –GTP aa-tRNA - EF-Tu –GTP Translocation Elongation empty

43 Copyright © 2013 Pearson Canada Inc a)30S subunit (blue) is shown in starting conformation after termination (outlined in red) to a fully rotated conformation seen during elongation (black outline). b)During transition to the fully rotated state, tRNAs shift from binding in A/A and P/P sites (30S/50S) to occupying hybrid sites A/P and P/E. c)Rotation in another plane can move the head domain of the 30S subunit as much as 14°toward the E site. A schematic view of ribosome subunit rotational motions View from the bottomSide view (step 3)

44 Translocation of peptidyl tRNA from the A site to the P site Copyright © 2013 Pearson Canada Inc At this point the E and P sites are occupied, but A is empty. As the deacylated tRNA is released from E, the A site gains high affinity and accepts the aminoacyl tRNA dictated by the next codon. A cycle of elongation is now complete. All is as it was at the start, except that now: 1.The polypeptide chain has grown by one residue. 2.The ribosome has moved along the mRNA by three nucleotide residues—one codon. 3.At least two molecules of GTP have been hydrolyzed. The whole process is repeated again and again until a termination signal is reached. (step 4) (step 5)

45 Termination of Translation Copyright © 2013 Pearson Canada Inc The completion of polypeptide synthesis is signaled by the translocation of one of the stop codons (UAA, UAG, UGA) into the A site. Because there are no tRNAs recognizing these codons under normal circumstances, termination of the chain does not involve binding of a tRNA. Instead, protein release factors (RFs) participate in the termination process.

46 Copyright © 2013 Pearson Canada Inc Termination of translation in prokaryotes RF1 : recognizes UAA & UAG RF2 : recognizes UAG & UGA RF3 : a GTPase stimulating the release process via GTP binding and hydrolysis

47 Interaction of RF2 with a UGA stop codon in the decoding center Copyright © 2013 Pearson Canada Inc The ribosome in complex with RF2. UGA is in magenta, and RF2 is in green, except for those parts interacting directly with the codon, which are shown in red. RF2 helical domains that move significantly are shown in color, with the extent of the movements (domains 1 and 3), in the same color scheme, shown below. UGA RF2

48 Suppression of nonsense mutations Copyright © 2013 Pearson Canada Inc The effects of nonsense mutations can sometimes be suppressed by suppressor mutations, in which a tRNA mutates to recognize a stop codon and inserts an amino acid instead.

49 A nonsense mutation can be overcome by an intergenic suppression mutation Copyright © 2013 Pearson Canada Inc A nonsense mutation in a protein- coding gene changes a codon for an amino acid into a stop codon, causing translation to terminate prematurely. Another mutation, in a tRNA gene, can circumvent the first mutation by altering the tRNA anticodon so that it will base-pair with the mutant mRNA. A functional protein is produced in this situation, even though suppression might not restore the original amino acid at that site.

50 Copyright © 2013 Pearson Canada Inc Some antibiotics that act by interfering with protein biosynthesis The translational machinery of eukaryotes is sufficiently different from that of prokaryotes that these antibiotics can be used safely in humans. Tetracycline would also inhibit eukaryotic translation; however, it cannot transverse the cell membrane of higher organisms.

51 Translation in eukaryotes Copyright © 2013 Pearson Canada Inc The mechanism for translating mRNA into protein in eukaryotic cells is basically the same as in prokaryotic cells. In eukaryotes, the ribosomes are larger and more complex, and virtually all mRNAs are monocistronic. In eukaryotes, translation initiation is more complex and requires more protein factors (eIFs) than in prokaryotes. The major differences from prokaryotic initiation are associated with cap binding and with the hunt for the first AUG.

52 Initiation of translation in eukaryotes Copyright © 2013 Pearson Canada Inc The 5’ end of a message is sensed not by a Shine-Dalgarno sequence, but by the 7-methylguanine cap. The N-terminal amino acid, inserted at the initiator AUG, is methionine (Met), not N-formylmethionine (fMet).

53 Inhibitors effective only in eukaryotes Copyright © 2013 Pearson Canada Inc Two important ones are diphtheria toxin and cycloheximide. eEF2 NAD + ADP-ribosylated o Diphtheria toxin is an enzyme, coded for by a bacteriophage that is lysogenic in the bacterium Corynebacterium diphtheriae. o The toxin catalyzes the synthesis of a derivative of a modified histidine in eEF2 using NAD +, thereby inactivating eEF2 and blocks protein synthesis. o Pure diphtheria toxin is one of the deadliest substance known. Cycloheximide inhibits the translocation activity in the eukaryotic ribosome and is often used in biochemical studies when processes must be studied in the absence of protein synthesis.

54 Rate of translation in prokaryotes Copyright © 2013 Pearson Canada Inc At 37 °C, an E. coli can synthesize a 300-residue polypeptide chain in ~20 seconds (15 codons per second). Recent studies with E. coli show that a ribosomal protein, NusE, interacts in the cell with an RNA polymerase component, NusG. Through this interaction, transcription and translation are physically coupled. Direct coupling of this type cannot occur in eukaryotic cells. (why not?)

55 Copyright © 2013 Pearson Canada Inc The growth of individual polypeptide chains does not account for the total rate of protein synthesis in the cell. Many ribosomes may be simultaneously translating a given message, observed as polyribosomes (also called polysomes ). Rate of translation in prokaryotes translation

56 Energetics of translation Copyright © 2013 Pearson Canada Inc The energy cost for this process is high. If we examine the individual steps in protein synthesis described earlier, we can make the following estimate of the total energy budget for synthesizing a protein of N residues: Translation is fast but energy-expensive. About four ATP equivalents are needed for each amino acid added.

57 The final stages in protein synthesis Copyright © 2013 Pearson Canada Inc Translation is immediately followed by various kinds of protein processing, including: o Chain folding (starts during translation and is nearly complete by the time the chain is released) o Covalent modification (some occur during translation, e.g. deformylation of N-fMet) o Directed transport (e.g. secretion, protein targeting to membrane or organelle location)

58 Covalent modification Copyright © 2013 Pearson Canada Inc Bacterial proteins that are destined for secretion (translocation across the cell membrane) are characterized by highly hydrophobic signal sequences or leader sequences in the N-terminal regions. After the protein has passed through the membrane, the leader sequence is cleaved off. cleave

59 A current model for protein secretion by prokaryotes Copyright © 2013 Pearson Canada Inc The new polypeptide chain (the pro-protein) complexes with SecB, which prevents complete folding during transport to the membrane. At the membrane an ATPase, SecA, drives translocation through the membrane with the aid of the SecYEG translocon. The SecYEG translocon is composed of SecE, SecY, and SecG, and forms a membrane pore. The leader sequence is then cleaved off the secreted protein by a membrane peptidase.

60 An outline of the mechanism of protein splicing Copyright © 2013 Pearson Canada Inc. A small number of proteins, mostly from single-cell organisms, undergo post-translational splicing, in a process comparable to RNA splicing Internal protein segment External protein (yielding the mature protein)

61 Protein targeting in eukaryotes Copyright © 2013 Pearson Canada Inc Proteins destined for the cytoplasm, nuclei, mitochondria, and chloroplasts are synthesized in the cytoplasm. Those destined for organelles have specific targeting sequences, which probably aid in membrane insertion, but they also interact with a particular class of chaperons (e.g. members of “heat-shock” Hsp70 family) Proteins destined for cell membranes, lysosomes, or export are synthesized on the rough endoplasmic reticulum, then modified and transported via the Golgi apparatus.

62 Transport of newly synthesized mitochondrial proteins into the matrix Copyright © 2013 Pearson Canada Inc Upper left, signal sequence of cytosolic Hsp70-bound protein inserts into import receptor of the TOM complex (TOM20) in the outer membrane. Hsp70 dissociation is coupled to ATP hydrolysis. Insertion of the protein into the outer membrane (via TOM22) puts signal sequence in position to interact with TIM complex (TIM23) in the inner membrane. Potential across the inner membrane drives protein into intermembrane space. Signal sequence is cleaved off by MPP (matrix processing peptidase). Mitochondrial Hsp70 binds to protein in the matrix and uses the energy of ATP hydrolysis to pull the rest of the protein through. T ranslocation of o uter m embrane T ranslocation of i nner m embrane Potential

63 Delivery of a protein, synthesized in cytoplasm, into the nucleus Copyright © 2013 Pearson Canada Inc A protein with a nuclear localization signal ( NLS ) binds to importin as a cargo. The NLS can be found anywhere within the polypeptide sequence, and is not removed after transport. The importin-cargo complex binds to a nuclear pore and passes through. Within the nucleus Ran-GTP binds to the importin-cargo complex and displaces the cargo. The resultant Ran-importin complex is returned to the cytoplasm, where Ran- bound GTP is hydrolyzed to GDP. Ran-GDP returns to the nucleus, and its bound GDP is exchanged for GTP (not shown). cargo Ra s-related n uclear protein Ran GEF

64 Proteins synthesized on the rough endoplasmic reticulum Copyright © 2013 Pearson Canada Inc Proteins destined for cell membranes, lysosomes, or export are synthesized on the r ough e ndoplasmic r eticulum ( RER ). Signal recognition particle In step 6, proteins that will remain in the ER have resistant signal peptides and thereby remain anchored to the RER membrane. cleavage Ducking protein

65 An actively translating and translocating eukaryotic ribosome-Sec61 complex Copyright © 2013 Pearson Canada Inc Protein-conducting channel (Sec61) Nascent chain Peptidyl-tRNA

66 Role of the Golgi complex Copyright © 2013 Pearson Canada Inc Note that vesicles bud off the RER and move to the cis face of the Golgi. Primary lysosomal vesicles bud from the trans portion of the Golgi. cis face Trans face

67 A schematic view of SNARE–pin fusion Copyright © 2013 Pearson Canada Inc Specific v-SNAREs (vesicle) and t-SNAREs (target) dictate interaction, and form coiled-coil structures. After fusion, these are broken up by the factor NSF, using the energy of ATP hydrolysis. By J.E Rothman et al. (2013 Nobel Prize in Physiology or Medicine)

68 The fate of proteins: programmed destruction Copyright © 2013 Pearson Canada Inc Proteins need to be selectively degraded to control the cellular function (e.g. regulatory proteins in the cell cycle, damaged or incorrectly synthesized proteins). Eukaryotic cells have two distinct methods for protein degradation. ○ The lysosome system —protein degradation occurs via lysosomes (vesicles filled with hydrolytic enzymes) ○ The cytosolic degradation system —protein degradation occurs in the cytosol, often involving the marker ubiquitin and/or a multicatalytic complex (the proteasome).

69 Formation of primary & secondary lysosomes: their role in digestive processes Copyright © 2013 Pearson Canada Inc The primary lysosomes that bud from the Golgi are esseentially bags of degradative enzymes. They can take several pathways. o Path A: Exocytosis —transport of enzymes to outside of cell. o Paths B and C: Phagocytosis — formation of phagic lysosomes for digesting organelles ( auto phagocytosis) or ingested matter ( hetero phagocytosis). o Path D: Autolysis —destruction of the cell itself (self-digestion). macrophage

70 Apoptosis Copyright © 2013 Pearson Canada Inc Apoptosis, the major form of programmed cell death, is triggered by either an extrinsic or intrinsic pathway. Both pathways initiate a series of proteolytic activations of procaspases to active caspases. ○The caspase name refers to the fact that the enzyme has an active-site cysteine residue, and attacks target proteins at specific aspartate residues (c-asp-ase). ○The caspase proteins are synthesized as procapases, which undergo proteolytic cleavage and dimerization, yielding enzymatically active α 2 β 2 heterotetramers. Tumor cells lose the ability to undergo apoptosis.

71 Extrinsic signaling pathway leading to apoptosis Copyright © 2013 Pearson Canada Inc The extrinsic pathway predominates during normal development. D eath- i nducing s ignaling c omplex inactive active (TNF)

72 Intrinsic signaling pathway leading to apoptosis Copyright © 2013 Pearson Canada Inc The intrinsic pathway is activated as a result of intracellular damage (e.g. DNA damage). A poptotic p rotease a ctivating f actor (interact with Mt outer membrane) hepamer


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