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Protein synthesis: methionly-tRNAi recognizes the AUG start codon Initiation factor Translation initiation usually occurs near the first AUG closest to the 5’ end of an mRNA eIF4A (helicase activity) →uses energy→ unwind RNA→complex move Kozak sequence: ACCAUGG UTR Initiator factor: IF • Initiation The inactive 40S and 60S subunits will bind to each other with high affinity to form inactive complex This is achieved (達到) by eIF3, which bind to the 40S subunit mRNA forms an preinitiation complex (also bound eIF1A, Met-tRNAi Met, eIF, GTP) with a ribosome; when eIF 2 phosphorylated → GDP/GTP exchange X→ translation X A number of initiation factors participate in the process. Cap sequence present at the 5’ end of the mRNA is recognized by eIF4 complex, eIF4 interaction with preinitation complex. Subsequently eIF3 is bound and cause the binding of small 40S subunit in the complexes (initiation complex) step 2→ slide or scan; eIF4A plus mRNA → helicase→open RNA secondary structure (ATP dependent) The 18S RNA present in the 40 S subunit is involved in binding the cap sequence eIF2 binds GTP and initiation tRNA, which recognize the start codon AUG → eIF2 GTP hydrolysis → irreversible step for further scanning. eIF4A unwind the RNA secondary structure by hydrolysis of ATP, 40S complex migrate down stream until it finds AUG start codon eIF5 hydorlysis a GTP →The large 60S subunit is then bound to the 40S subunit It is accompanied by the dissociation of several initiation factor and GDP The formation of the initiation complex is now completed Ribosome complex is able to translate During chain elongation each incoming aminoacyltRNA move through three ribosomal sites Elongation factor (EFs): help ribosome move and tRNA move Translocation: ribosome move correct Conformational change Peptidyltransf erase reaction by large rRNA Help move ribosome contains two sites where the tRNAs can bind to the mRNA. P (peptidyl) site allows the binding of the initiation tRNA to the AUG start codon. The A (aminoacyl) site covers the second codon of the gene and the first is unoccupied On the other side of the P site is the exit (E) site where empty tRNA is released The elongation begins after the corresponding aminoacyl-tRNA occupies the A site by forming base pairs with the second codon Two elongation factors (eEF) play an important role. EF1 and EF2 eEF1α binds GTP and guides the corresponding aminoacyl-tRNA to the A site, during which GTP is hydrolized to GDP and P. 1 correctly base-pair → hydrolysis GTP → conformational change → tight bind aminoacyl-tRNA in A site and release EF1-GDP Peptide bond formation by large rRNA (peptidlytransferase reaction) The cleavage of the energy-rich anhydride bond in GTP enables the aminoacyl-tRNA to bind to codon at the A site Afterward the GDP still bound to eEF1α, is exchange for GTP as mediated by the eEF1βγ, can recycle The eEF1 α-GTP is now ready for the next cycle Subsequently a peptide linkage is form between the carboxyl group of methionine and the amino group of amino acid of the tRNA bound to A site Peptidyl transferase catalyzing the reaction. It facilitates the N-nucleophilic attack on the carboxyl group, whereby the peptide bond is formed with the released of water. Accompanied by the hydrolysis of one molecule GTP to form GDP and Pi, the eEF2 facilitates the translocation of the ribosome along the mRNA to three bases downstream Free tRNA arrives at site E is released, and tRNA loaded with the peptide now occupies the P Site The third aminoacyl-tRNA binds to the vacant A site and a further elongation An RNA-RNA hybrid of only three base pairs is not stable for normal physiological condition. Multiple interactions between the large and small rRNAs and general domains of tRNAs can stabilized the tRNA in the A and P site. E. Coli 70S ribosomes 2 Translation is terminated by release factors when a stop codon is reached Release factor RF: In eukaryote eRF1 like tRNA, can bind to A site of ribosome eRF3 is GTP binding protein Promote cleavage of the peptidyl-tRNA, and releasing the protein chain In bacterial RF1 and RF2 like eRF1 RF 3 GTP bind factor Chaperone protect protein and help folding GTPase When A site finally binds to a stop codon (UGA, UAG, UAA) Stop codons bind eRF accompanied by hydrolysis GTP to form GDP and P Binding of eRF to the stop codon alters the specificity the peptidyl transferase Water instead amino acid is now the acceptor for the peptide chain Protein released from the tRNA Protein synthesis-4 Termination of translation When the ribosome reaches a stop codon in the A site, one of three releasing factors and initiate hydrolysis of the peptide chain from the tRNA in the P site – RF-1 recognizes UAA and UAG – RF-2 recognizes UAA and UGA – RF-3 binds GTP and enhances the effects of RF-1 and RF-2 Polysomes and rapid ribosome recycling increase the efficiency of translation Polysomes and recycling increase translation Eukarotic mRNA in circular form stabilized by interactions between protein bound at the 3’ and 5’ ends Poly A binding protein (PABPI), interact with both mRNA poly A and eIF4g Two ends is very close together, then ribosome subunit easy to bind. 3 The Synthesis of Protein Polyribosomes = A cluster of ribosomes simultaneously translating an mRNA molecule Polyribosomes are found in both prokaryotes and eukaryotes Protein-protein and protein –mRNA interactions form a bridge. Purified poly(A)-binding protein I, eIF4E and eIF4G , mRNA formed a circular structure Life cycle of an mRNA-2 The biological activity of proteins depends on a precise folding of the polypeptide chain into 3-D conformation Some proteins must undergo post-translational modification before they become fully functional DNA replication: DNA-directed DNA polymerases Semi-conservative 5’ – 3’ Replication forks Uni- or Bi- directional Semi-discontinuous Primers 4 DNA replication is semiconservative mechanism DNA polymerase require a primer to initiate replication DNA replication direction: 5’ to 3’ DNA polymerase need a primer to initiation. Action of DNA polymerase. DNA is elongated in its 5′ → 3′ direction. Helicase : open DNA double strand Replication origin DNA polymerase Primase: provide a short primer Replication fork Topoisomerase I: release the local unwinding of DNA produces torsional stress (扭力; supercoil) DNA ligase Single-stranded binding proteins Leading strand Lagging strand Helicase: separates the two DNA strands, starting at replication origins (rich in A-T base pairs) RNA primase: inserts a starter of RNA nucleotides at the initiation point DNA polymerase binds a complementary leading strand of DNA nucleotides starting at the 3’end of the RNA primer Exonuclease removes RNA primer, which are replace with DNA nucleotides by DNA polymerase 5 Two strands are anti-parallel & DNA, polymerase synthesizes 5’ TO 3’ DNA synthesis is discontinuous on the lagging strand but continuous on the leading strand (Okazaki et al 1968). The short DNA fragments on lagging strand are called Okazaki fragments. DNA polymerase requires a primers so each Okazaki fragment must begin with a primer. How are primers synthesized? First primer (starts strand synthesis) and primers for each Okazaki fragment Primases starting nascent DNA chains Primases synthesize short RNA (or RNA/DNA) oligonucleotides that act as primers for DNA polymerase. Can initiate synthesis on ssDNA de novo (no 3’=OH needed). Usually part of protein complex or need specific interactions with other replication proteins for efficient primer synthesis. Most primases start synthesis at a random sites; do not synthesize primers with a specific sequence. Homotrimeric Heterotrimeric protein, maintain the template in a uniform conformation for DNA polymerase 1.Like Helicase open DNA (unwind) 2.RPA (heterotrimeric protein) bind single DNA, Single-stranded binding proteins 3.Leading strand synthesis by DNA polymerase s, PCNA and Rfc (replication factor) complex 4.Lagging strand synthesis by pol σ→Okazaki fragment 5. PCNA-Rfc-pol σ complex process each Okazaki fragment Proliferating cell nuclear antigen: prevent loss complex dissociating from DNA Maintains the template in a uniform conformatio Replication protein A DNA helicases - Separation of the Watson/Crick helix DNA helicases utilize energy of ATP hydrolysis to cause disruption of hydrogen bonds in the double helix. Helicases are necessary for movement of a replication fork. In E. coli, primary replicative helicase is dnaB Helicases function by moving along ssDNA in one direction disrupting hydrogen bonds as they move. Both 5’ to 3’ and 3’ to 5’ helicases exist. Nomenclature - direction of helicase movement is defined on the strand the helicase binds. (A 5’ to 3’ helicase isshown at right). Single-stranded DNA binding proteins (SSBs) bind tightly to ssDNA. SSBs prevent formation of secondary structure, renaturation of ssDNA and non-specific interactions on ssDNA. – SSBs usually bind cooperatively. – SSBs usually interact with other replication proteins; these interactions promote efficient replication 6 Role of Topoisomerases SSBs bind tightly to ssDNA. – SSBs prevent formation of secondary structure, renaturation of ssDNA and non-specific interactions on ssDNA. – SSBs usually bind cooperatively. – SSBs usually interact with other replication proteins; these interactions promote efficient replication Replication fork → move →supercoil During DNA replication Topoisomerases act to release the links between the parental DNA strands both during replication (swiveling轉環) or after replication (decatenation去除連銷). DNA ligases form phosphodiester bonds; join strands of DNA Topoisomerases Type I - change L by multiples of 1 by causing a transient ssDNA break. Type II - change L by multiples to 2 by causing a transient dsDNA break. Topoisomerases function by forming a covalent intermediate with the transiently broken end(s) of the DNA. Almost all topoisomerases relax both positively and negatively supercoiled DNA. Topoisomerase I lagging strand is used in discontinuous synthesis forms Okazaki fragments fragments joined by DNA ligase Must supply a primer (i.e. 3’-OH) to start DNA synthesis This is the function of primase which makes RNA primers Must ‘seal’ the DNA fragments made on the lagging strand template This is the function of DNA ligase After DNA is synthesized, RNA primer is being degraded and replaced by DNA (strand replacement synthesis). 7 The Okazaki fragment In prokaryotes, the leading and lagging strand DNA replication machines are associated. Okazaki fragments are the short DNA fragments produced during lagging strand DNA synthesis. They will be ligated together by ligase shortly after completion. Prokaryotes like E. coli has Okazaki fragment of 1000~2000 nucleotides long while eukaryotes like us has shorter Okazaki fragments (100~200 nucleotides) DNA replication generally occurs bi-directionally form each origin Mapping Using Electron-microscope – Isolate partially replicated DNA (replication intermediates). Enrich using di-deoxynucleotides or density labeling. – Compare location of replication bubble for a number of molecules (many) – Orientation of DNA! Need reference point. Usually a restriction site. – Very small bubbles identify location of origin. – Movement of ends indicates number of active forks. Eukaryotic chromosomal DNA contain mutiple replication origins separated by tens to hundreds of kilobases. ORC: origin recognition complex (6 subunits) combine with other factor (such as hexameric helicases) to start replication. 8 1. 2. 3. 4. 5. 6. 7. Using energy, ATP hydrolysis → single DNA and bound RPA Primase and pol α complex synthesis short primer PCNA-Rfc-Pol σ complex replace the Primase and pol α complex → generate leading strand helicase unwind the parental strands, and RPA bind to newly single strand Coordination of the leading and lagging strand synthesis-5 PCNA-Rfc-Pol σ complex synthesis DNA Primase and pol α complex synthesis short primer for lagging strand PCNA-Rfc-Pol σ complex replace the Primase and pol α complex → extend the lagging strand –Okazaki fragments. It eventually ligated to the 5’ends of the leading strands. Rfc: replication factor c Mutation 1. Spontaneous errors in DNA replication (10 -7) 2. A consequence of the damaging effects of physical or chemical mutagens on DNA DNA repair and recombination Several mechanisms can prevent it (repair system). 1. DNA polymerase proof reading 2. Base excision repair; T-G mismatch repair (one base) 3. Mismatch excision repairs (several base) 4. Nucleotide excision repair (transcription-coupled repair) 5. Recombination to repair double strand breaks in DNA 9 DNA polymerase introduce copying errors and also correct them DNA polymerase is the first line of defense in preventing mutation. It can proofreading. In E. coli about 1/104 happen, however, only about 1/109 nucleotides incorporated into growing strand. Proofreading depends on 3’-5’ exonuclease activity of some DNA polymerase. Uncorrected base-paring → polymerase stop → transfer 3’ end of growing strand → to its exonuclease site → remove E. Coli only has one type DNA polyermase, in eukaryotic DNA δ and ε, used for proofreading activity. I Proofreading by DNA polymerase Bind to single strand template 3nm Uncorrected base-pairing in 3’ end → melting of newly formed end of the duplex→ polymerase stop → transfer to exonuclease site (exo) Polymerase catalytic activity Chemical and radiation damage to DNA can lead to mutations In normal cell, many chemical reaction (lipid oxidation, peroxisome, mitochondria…..) always work. Environmental factor always damage to DNA. Many spontaneous mutations are point mutations. Most frequent point mutation are deaminiation: cytosine (C) base convent to uracil (U) base. Common modified is 5-methycytosine convent to thymine via deamination. Other environmental factor: UV, ionizing radiation……. DNA undergoes damage spontaneously from hydrolysis and deamination > unnatural sites Deamination: C > U (pairing with A) A > hypoxanthine (with C) G > xanthine (with C) 5-mC > T Depurination: common Depyrimidination (脱嘧啶作用) : rare 10 Point mutation: a single base change Depurination and deamination Transition : Purine or pyrimidine is replaced by the other A↔G T ↔C Transversion : a purine is replaced by a pyrimidine or vice versa A ↔T or C T ↔ A or G G ↔T or C C ↔ A or G > Genetic polymorphism Spontaneous Alterations of nucleotides Red: oxidative damage; blue: hydrolytic attack; green: uncontrolled methylation High-Fidelity DNA excision-repair systems recognized and repair damage Excision-repair systems: high homologs of key bacteria protein exist in eukaryotes; similar manner process: segment of the damaged DNA is excised → gap → filled by DNA polymerase → ligase → repair ok In normal, most common point mutation is C to T. using base excision repair system can repair it. Other mutation such as C to U or 5-methyl C to T also using the same system. 11 II Base excision repair of a T.G mismatch RECOGNIZED Human cells contain a battery of glycosylases, is specific for a different set of chemically modified DNA base. DNA glycosylases Base excision repair system Base excision repair system works primarily on modifications caused by endogenous agents At least 8 DNA glcosylases are present in mammalian cells DNA glycosylases remove mismatched or abnormal bases cleaves N-glycosylic bond AP endonuclease Apurinic exdonuclease I cleaves apurinic or apyrimidinic site AP endonuclease cleaves 5’ to AP site AP lyase cleaves 3’ to AP site DNA polymerase 3’→5’ exonuclease activity & polymerase activity Mismatch excision repairs other mismatches and small insertions and deletions III Another DNA repair systems, is also conserved from bacteria to human Eliminates base-pair mismatches, insertions or deletions or few nucleotides that are accidentally produced by DNA polymerase. Mismatch excision repair: determining normal and mutant DNA → repair latter. Bacteria Eukaryotes MutS MSH1-6 MutL MLH1, PMS1-2 Homolog to bacteria MutS 2 and 6 Mismatch Repair in Human Cells MSH2 and MSH6 bind to mismatchcontaining DNA and distinguish between the template and newly synthesized strand Homolog to bacteria MutL MLH1 nicks the newly synthesized DNA and an exonuclease removes the mismatched base The gap is filled in by DNA polymerase and DNA ligase Defective mismatch repair is the primary cause of certain types of human cancers from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-37 12 VI Nucleotide excision repairs chemical adducts that distort normal DNA shape When chemical modified bases → nucleotide excision repair system six core factors encompassing 15 to 18 polypeptide chains for excision, plus repair synthesis and ligation Certain protein →slide along double stranded DNA → search bulges or irregularities shape → endonucleases activity → repair Nucleotide excision repair also called transcription-coupled repair Nucleotide excision repair in human cells Shared subunits in transcription and DNA repair at the same time. DNA damage in higher eukaryotes is repaired at a much faster rate in regions of the genome being actively transcribed than in nontranscribed regions Nucleotide Excision repair enzymes cleave damaged DNA on either side of the lesion XP-G + RAP → unwind and distabilize Transcription factor endonuclease 24-32 base endonuclease Transcription-coupled repair: nucleotide excision repair (NER) system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template 13 Two systems utilize recombination to repair doublestrand breaks in DNA Emergency DNA Repair for Double helix break Ionizating radiation or cancer drugs → double strand break → nonhomologous end joining or homologous recombination → repair Nonhomologous end joining Ku and DNA-dependent protein kinase 1. Complex bind to break DNA end; 2. removal of a few base by nucleases activity 3. ligate Homologous recombination can repair DNA damage and generate genetic diversity Homologous recombination DNA recombination: exchange of strands between separated DNA molecules Meiotic recombination Recombination has DNA repair mechanism and generated genetic diversity. Generate genetic diversity among the individuals of a species by causing the exchange of large regions of chromosomes between the maternal and paternal pair of homologous chromosomes during the cellular division the generates germ cells Meiotic recombination 14 Repair of a collapsed replication fork 倒塌 RecA/Rad 51 catalyzed invasion of a duplex DNA by a single stranded complement of one of the strands is key to the recombination process. Moreover, no base air are lost or gained in this process, called strand invasion If do not repair, generally death to at least one daughter cell Formed double strand break RecA and Rad51 are homologous protein; It bind to single strand DNA → → created fork collapse → invading 入侵 another single strand → formed perfectly complementary hybridization Dark red is invading strand ATP Double strand DNA break repair by homologous recombination 15 heteroduplex: is any region of doublestranded nucleic acid (DNA, RNA), where the two strands come from two different original molecules. Mismatch repair system If very complementary Gene Conversion 16 Holliday Model of recombination: resolution 回覆 (1964) Non-crossover Crossover event Enzyme vs. DNA replication DNA replication is bi-direction 17 Prokaryote-Eukaryote Differences Viruses: parasites of the cellular genetic system Most viral host ranges are narrow Viruses cant not reproduced by themselves (no life without host) RNA virus: replicate in the host cell cytoplasm DNA virus: replicate in the host cell nucleus Viral genomes has single or double stranded Virion: entire infectious virus particle, consists nuclei acid and shell of protein Bacteriophage (phage): infect only bacteria Head Animal virus or plant virus Tail DNA sheath Tail fiber Physical Characteristics Genetic Material Nucleic acid RNA (ssRNA, dsRNA, segmented) DNA (ssDNA, dsDNA) Protein coat (subunit structure) Nucleoprotein Capsid Capsomeres, Geometrical constraints Envelope (some) 80 × 225 nm Figure 18.4d VIRUS STRUCTURE Basic rules of virus architecture, structure, and assembly are the same for all families Some structures are much more complex than others, and require complex assembly and dissassembly The capsid (coat) protein is the basic unit of structure; functions that may be fulfilled by the capsid protein are to: – – – – – – Protect viral nucleic acid Interact specifically with the viral nucleic acid for packaging Interact with vector for specific transmission Interact with host receptors for entry to cell Allow for release of nucleic acid upon entry into new cell Assist in processes of viral and/or host gene regulation 50 nm (d) Bacteriophage T4 Nucleoprotein must be stable but dissociatable Capsid is held together by non-covalent, reversible bonds: hydrophobic, salt, hydrogen bonds Capsid is a polymer of identical subunits Terms: – Capsid = protein coat – Structural unit = protein subunit – Nucleocapsid = nucleic acid + protein – Virion = virus particle Capsid proteins are compactly folded proteins which: – Fold only one way, and robustly – Vary in size, generally 50-350 aa residues – Have identifiable domains – Can be described topologically; similar topological features do not imply evolutionary relationships 18 Virus Structure – 1. Helical: single coat protein, tobacco mosaic virus 2. Icosahedron: 20 faces There are two major structures of viruses called the naked nucleocapsid virus and the enveloped virus. Helical symmetry A NAKED virus. The red balls represent the protein subunits that make up the protective covering around the viral genome (DNA in the case). These subunits are called CAPSOMERES and the entire protein coat is called the CAPSID An ENVELOPED virus. Enveloped viruses have a lipid-based membrane surrounding the protein capsid. This envelope is partly composed of the cell membrane within which the virus replicated, and it contains proteins and carbohydrates. Some of the proteins are from the host cell and some are from the virus Coat protein Tobacco mosaic virus is typical, well-studied example Each particle contains only a single molecule of RNA (6395 nucleotide residues) and 2130 copies of the coat protein subunit (158 amino acid residues) TMV protein subunits + nucleic acid will self-assemble in vitro in an energyindependent fashion Self-assembly also occurs in the absence of RNA Figure 18.4a, b Capsomere of capsid RNA Capsomere DNA Glycoprotein 18 × 250 mm 70–90 nm (diameter) 20 nm (a) Tobacco mosaic virus 50 nm (b) Adenoviruses RNA Function of the capsid/envelope Protect nucleic acid from the host’s acid- and proteindigesting enzymes Assist in binding and penetrating host cell Stimulate the host’s immune system 小兒麻痺 Monkey DNA Animal RNA Plant RNA 二十面體 TMV 19 SV40 structure Viral capsids are regular arrays of one or a few types of protein Capsid (protein coat): nucleic acid of a virion is enclosed, composed of multiple copies of one protein or few different protein. Nucleocapsid: a capsid plus the enclosed nucleic acid, protect functions; Two structure: Envelope: some vriuses, symmetrically arranged nucleocapsid is covered by an external membrane (envelope), which consists mainly of a phospholipid but also contains one or two types of virus-encoded glycoproteins. Enable pleomorphic (多形性) shape of the virus – Spherical (球形) – Filamentous (絲形) Viral protein spikes protrube Influenza 20 Lytic viral growth cycles lead to death of host cell E coil phage dd DNA 1.Adsorption 2.Penetration 3.Replication 4.Assembly 5.Release Plaque assay Clone: all the progeny birions in a plaque are derived from a single parental virus Degrade the host cell DNA → provide nucleotides for synthesis viral DNA Plate → seeding host cell → virus add → infect host cell → host cell lysis → plaque Capsid & assembly protein Lytic virus Viruses vs. life cycle Has envelope Viral Reproduction I ssRNA Bacteriophages are viruses that infect bacteria. They reproduce by: 子代 a) Lytic cycle Viral RNA polymerase replicated RNA H+ Induced viral glycoprotein conformational change b) Lysogenic cycle Fusion of viral envelope with endosomal lipid bilayer membrane and release of the nucleocapsid into cytosol 21 Viral DNA is integrated into host cell genome in some nonlytic viral growth cycles Progeny (後代) virions of enveloped viruses are released by budding from infected cells Some viruses, nonlytic association with host cell (not kill) is called temperate phages Prophage: integrated into the host cell chromosomes rather than being replicated Lysogeny: Instead of destroying host to produce virus progeny, the viral genome remains within the host cell and replicates with the bacterial chromosome. This relationship between phage and host is called lysogeny viral DNA is integrated into the hose cell genome in some nonlytic viral growth cycles Retroviruses • Such as HIV, use the enzyme reverse transcriptase – To copy their RNA genome into DNA, which can then be integrated into the host genome as a provirus Glycoprotein The lysogenic cycle – Replicates the phage genome without destroying the host Temperate phages – Are capable of using both the lytic and lysogenic cycles of reproduction Prophage: integrated viral DNA 1. Viral envelope Capsid 2. 3. Reverse transcriptase Figure 18.9 RNA (two identical strands) Viral glycoprotein in envelop interact with specific hose cell membrane → entry nucleocapsid into cytoplasm Viral reverse transcriptase and protein → copy the ssRNA to ds DNA→ ds DNA → transport into the nucleus → integrated HOST chromosomal DNA → leading to provirus two & translation Retroviral life cycle 22 HIV HIV Membrane of white blood cell 1 The virus fuses with the cell’s plasma membrane. The capsid proteins are removed, releasing the viral proteins and RNA. 2 Reverse transcriptase catalyzes the synthesis of a DNA strand complementary to the viral RNA. HOST CELL 3 Reverse transcriptase catalyzes the synthesis of a second DNA strand complementary to the first. Reverse transcriptase Viral RNA RNA-DNA hybrid 4 The double-stranded DNA is incorporated as a provirus into the cell’s DNA. 0.25 µm HIV entering a cell DNA NUCLEUS Chromosomal DNA end Provirus 5 Proviral genes are transcribed into RNA molecules, which serve as genomes for the next viral generation and as mRNAs for translation into viral proteins. RNA genome for the next viral generation mRNA 6 The viral proteins include cap proteins and reverse transcriptas (made in the cytosol) and envelo glycoproteins (made in the ER). Figure 18.10 9 New viruses bud New HIV leaving a cell off from the host cell. 8 Capsids are assembled around viral genomes and reverse transcriptase molecules. 7 Vesicles transport the glycoproteins from the ER to the cell’s plasma membrane. 23