Exam 2: Making Protein

Bio 230w Exam 2 Making Protein Explain how mRNA is translated into different locations (cytoplasm vs. ER) and how this impacts final protein localization (cytoplasm vs. export vs. membrane). Translated into cytoplasm ? occurs if protein codes for it supposed to stay in the cytoplasm, nucleus, or other organelles like mitochondria Ribosomes Translated into ER ? protein needs to be secreted out of the cell, cell membrane, or lysosomes Ribosomes bound on the ER Impacts final protein localization: Cytoplasm ? function within the cell Export ? packaged into vesicles, sent to Golgi, and released out of the cell Membrane ? embedded in the cell membrane and play roles in signaling and transport Compare and contrast the route of a cytoplasmic protein vs an integral membrane protein vs an exported protein Cytoplasmic protein Translation ? in the cytoplasm Localization ? remain in cytoplasm, nucleus, and other organelles Function ? work in the cell Integral membrane protein Translation ? starts in cyto but moves to ER Localization ? embedded in cell membrane Function ? cell signaling, transport, interaction with the environment Exported protein Translation ? starts in cyto but moves to ER Localization ? sent out of the cell through a secretory pathway (ER to Golgi..) Function ? outside the cell like hormones and enzymes Describe the structure and the role of the translocon, Sec 61, in the localization of proteins. Translocon = protein channel embedded in the membrane of the ER Sec 61 = made up of proteins that form a channel through which newly synthesized proteins can enter ER. Made up of a pore Role in localization of proteins: Secreted proteins = ribosome make the protein and the signal sequence directs it to Sec 61 and the rotein passes through translocation into ER to get packaged for export Membrane proteins = translocon opens sideways allowing part of the protein to enter the membrane instead of passing through Describe the role of the amino acid side chain in the function of proteins. Play a role in how the proteins are folded by hydrogen bonds, ionic bonds, and hydrophobic interactions The amino acids that span the hydrophobic core of the membrane are nonpolar, which means that they have nonpolar side chains that repel water Describe the role of a peptide bond. The bond that connects monomers to a polymer (polypeptide) Holds the protein together by connecting the amino acids into a polypeptide Explain the importance of R-groups or side chains in protein function. The r-groups determine the shape, structure, and function of the protein The r-groups make each amino acid different from the other Compare and contrast a DNA polymer and a polypeptide. DNA Polymer = composed of nucleotides, double helix, stores genetic information, found in the nucleus, stores genetic info DNA Polypeptide = composed of amino acids, linear chain, performs various functions, found throughout the cell, carries out cellular functions Similar ? Both polymers, are made up of long chains and have specific functions Describe the structure of tRNA and how its structure contributes to function. Structure 3D L shape, typically 70-90 nucleotides long Anticodon Loop = set of 3 nucleotides which pairs with complementary codon mRNA @Amino Acid Attachment Site = located at 3’ end, specific amino acid attach Function Anticodon = allows tRNA to recognize and bind appropriate codon to mRNA during translation AAAS = specific amino acids are added to the growing polypeptide chain, ensuring the correct amino acid is added based on mRNA codon Identify the states where G-proteins are activated and inactivated. ALL bind and eventually hydrolyze to the GTP Activated = bound to GTP Inactivated = bound to GDP Explain the role of G-proteins in the elongation process of translation. Ensure that the correct amino acids are added to the growing polypeptide chains Elongation factors bind to the charged tRNA and GTP Then deliver the correct amino acid to the ribosomes A site, where anticodon of the tRNA pairs with the codon on the mRNA GTP is hydrolyzed into GDP releasing energy, allowing tRNA to be placed on ribosome GPD is recycled back into GTP Describe the structure of a ribosome and how its structure contributes to its function (eg. ribozyme function, A-site, P-site, E-site). Large Subunit A-site = tRNA carrying amino acid enters ribosome and binds to mRNA codon P-site = tRNA binds that hold growing polypeptide chain E-site = After aa is added the tRNA exits the ribosome Compare and contrast the function of different non-coding RNAs we have discussed in class. rRNA and tRNA = protein synthesis rRNA – forms the core structure of the ribosome tRNA – transports amino acids to the ribosome, ensuring correct amino acids are added tothe growing polypeptide chain miRNA, siRNA, and incRNA = gene regulation miRNA – regulates gene expression by binding to complementary mRNA sequence siRNA – binds and promotes degradation of specific mRNA molecules lncRNA – regulation of gene expression at various levels Outline the factors (proteins) and the sequence of events involved in the initiation of translation. Initiation factors – assemble the translation machinery mRNA binding – small ribosomal subunit attacked the mRNA near the 5’ end Initiator tRNA – specialized tRNA carries the first amino acid that binds to the start codon on the mRNA Large ribosomal subunit – after initiator tRNA is in place the LRS joins the complex forming a complete ribosome (hydrolysis of GTP to GDP) Initiation complex formation – the initiator tRNA is positioned at the P site and the A site is ready for the next tRNA carrying amino acid Analyze the interaction of codons and anticodons and match them to the appropriate amino acid using the provided genetic code. Codon – sequence of 3 nucleotides on the mRNA that codes for amino acids Anticodon – sequence of 3 nucleotides on the tRNA complementary to mRNA Describe how the genetic code is redundant and how it affects codon: anticodon pairing (wobble). Redundancy = multiple codons encoded for the same amino acid Multiple codons for one amino acid, amino acids are specified by more than one codon Wobble = the first two base pairs are strict but the third base codon is flexible Outline the major steps of translation (initiation, elongation, termination) using appropriate keywords. Initiation mRNA binding – mRNA attached to small ribosomal subunit Initiator tRNA – tRNA carrying methionine binds to start codon Ribosome assembly –large ribosomal subunit joins forming initiation complex Elgonation Codon recognition – tRNA with complementary anticodon pairs with the next mRNA codon Peptide bond formation – amino acids are linked by peptide bonds Translocation – ribosome moves along mRNA, shifting tRNA from A to P site Termination Stop codon – ribosome encounters stop codon Release factors – release factors bind to stop codon Disassembly – all parts separate Describe the different levels of protein structure (primary, secondary, etc…). Primary – sequence of amino acids Secondary – alpha helices and beta sheets (folding patterns of the protein chain, AE is spiral shape and BS are pleated) Tertiary – 3D shape (formed by the interaction between side chains of amino acids) Quaternary – multiple polypeptide chains (two or more polypeptides come together) Explain which interactions dictate the levels of protein structure. Primary–peptide bonds (amino acids linked) Secondary–hydrogen bonds (between the backbone of polypeptides) Tertiary – hydrogen, ionic, hydrophobic, disulfide Quaternary – non-covalent and disulfide Compare and contrast structural motifs (super-secondary structure) and domains. Structural motifs Short recurring sequences of secondary structures that form patterns. Function in structural roles within protein Domains Larger distinct structural units that can fold independently. Function with distinct functions Compare and contrast and identify the 3-dimensional structures of different proteins using domain box diagrams, ribbon diagrams, and ball and stick diagrams Box Protein domains are shown as a box, highlighting how different domains are arranged Ribbon 3D structure of proteins that use ribbons to represent helices and sheets. Shows folding Ball and stick Atoms as balls and chemical bonds are as sticks. Highlights the arrangement of proteins Key Words Polypeptide (Amino acid, side chain, peptide bond, N-terminus/C-terminus) Amino Acid = building block of protein Side chain = the unique r group Peptide bond = bonds linking amino acids N-terminus/C-terminus = ends of polypeptide chains (N amine group, C carboxyl group) Cotranslational Translocation (Translocon, signal peptide, SRP - signal recognition particle/peptide) Translocon = channel in ER through polypeptide passes Signal peptide = short amino acids diffraction ribosome to ER SRP = binds to signal peptide and halts translation until ribosome stays at ER Ribosome (large subunit, small subunit, E, P, A sites, ribozyme) Large subunit = peptide bonds form Small subunit = binds to mRNA E,P,A sites = where the tRNA binds (exit, peptidyl, aminoacyl) Riboxzyme = RNA molecule within ribosome that starts peptide bond formation tRNA (anticodon) The sequence of 3 nucleotides on the tRNA complementary to mRNA Small G-protein (active vs. Inactive) Active = bound to GTP Inactive = bound to GDP Canonical binding vs. non-canonical binding Canonical = standard, expected interactions Non-canonical = non-standard interactions, unusual pairings or modifications Initiation (start codon, 5’ mRNA cap) Start codon = the AUG codon where translation begins 5’ mRNA cap = modified G nucleotides added to 5’ end of mRNA for protection and initiation of translation Elongation (tRNA, codon, anticodon, 5’ end of RNA, 3’ end of RNA, ) tRNA = transfers amino acids to the ribosome for synthesis Codon = sequence of 4 nucleotides on mRNA encoding for amino acid Anticodon = complementary to mRNA codon, found on tRNA 5’ end = the beginning of mRNA 3’ end = end of mRNA Termination (Termination: stop codon, release factor, GTP hydrolysis) Stop codon = end of translation Release factor = proteins that bind to stop codon and release synthesizes polypeptide GTP hydrolysis = provides energy for the release of polypeptide Domain vs. structural motif Domain = large, functional unit that folds independently Motif = smaller, recurring pattern with a protein Hydrophobic interactions Nonpolar amino acid side chains that cluster together to avoid water stabilize the protein structure Membrane Tracking Define lipidation and its role in membrane proteins. Lipid molecules are covalently attached to proteins Role Anchor proteins to cell membranes Influence protein stability, activity, and interactions Signal transduction pathways Review how the location of protein translation and/or post-translational modifications contribute to the final protein location including inner membrane vs. outer membrane leaflet. Protein translation Cytoplasm = destinies for the cytosol, nucleus, or mitochondria ER = secretion, incorporation into the plasma membrane Post-translational modifications Glycosylation = adding sugar molecule to protein occurring in ER and Golgi Lipidation = covalent attachment of lipid groups that helps anchor proteins Phosphorylation = addition of phosphate groups that regular protein activity Proteolytic cleavage = removal of specific peptide segments Final Protein Location Inner leaflet = proteins involved in intracellular signaling Outer leaflet = proteins exposed to the extracellular environment ER = integrated into the membrane with specific orientations Golfi = further modifications and starting Analyze how changing the structure of a membrane protein (hydrophobic vs. hydrophilic regions) may alter its function or interaction with a lipid bilayer. Hydrophobic – residues in lipid biliary interacting with hydrophobic tails. Changes in this region can affect the protein's ability to embed in the membrane Hydrophilic – exposed to aqueous environment. Alterations to these regions can affect proteins' interaction with other molecules Analyze the characteristics of fatty acids that impact membrane fluidity using a temperature vs. membrane conversion chart Lower fluidity Saturated fatty acids Longer tails, less fluid, stronger Van Der Waal forces, more malleable Membrane fluid at higher temps Higher fluidity Unsaturated fatty acids Short tails, kinks (double bonds), weaker Van Der Waal forces Membrane fluid at lower temps Define anterograde vs. retrograde transport in a cell. anterograde – from inner to outer Retrograde – from outer to inner Describe the role of coat proteins in vesicle formation. Helping shape the membrane into a bud and facilitate the selection of cargo to be transported Bind to the membrane and cause it to curve forming a bud (vesicle formation) The coat proteins pinch off the vesicle from the membrane and release Describe the structure of clathrin (and a triskelion). Formation of coated vesicles during endocytosis Composed of 3 clathrin heavy chains (long and flexible forming an arm) and 3 clathrin light chains (smaller associated with heavy chains) A concave center that sucks membrane out Failed = vesicle formation would not work properly Define the role of dynamin in endocytosis. Final stages of endocytosis, separation of vesicles from the plasma membrane “Pinch off” vesicles from the other organelles by wrapping around the stem of the forming vesicle Failed = vesicles would not be able to separate from the membrane Compare and contrast vesicle budding (fission) vs. fusion. Fission Initiates transport, proteins involved are coat proteins and dynamin, encapsulates cargo into vesicles Fusion Completes transport, SNARE proteins involved deliver cargo to the target Describe the general pattern of cytoskeleton location as related to vesicle transport. Actin near the membrane and microtubules near the nucleus Actin = supports short-distance transport and vesicle docking Microtubules = serve as highways for long-distance transport of vesicles Identify where a G-protein is active in a cellular process, like vesicle transport. Active in regulating the formation, movement, and fusion of vesicles Involved in recruiting coat proteins Regulate motor proteins that transport vesicles along actin and microtubules SNARE complex assembly Outline the role of small G-proteins and SNARE proteins in vesicle fusion and analyze what may occur if proteins fail to function. Small G-proteins Rab G-proteins Function as molecular switches cycling between active GTP-bound state and inactive GDP-bound state Regulate vesicle trafficking SNARE proteins vSNARE = vesicle membrane tSNARE = trager memberrane Consequences: Small g-protein = vehicles may not correctly tether and dock at their target membranes leading to impaired vesicle transport SNARE = vesicle fusion with target membrane may not occur Review major topics from Sci Lit Week 4 (Ricin). Composed of two chains: A chain = starts protein synthesis by inactivating ribosomes B Chain = binds to carbo on cell surface Enters cell via endocytosis and the b chain binds to carb allowing a chain to enter and disrupt protein synthesis Key Words Membrane proteins (integral vs. peripheral), lipid-anchor Integral – embedded within the bilayer Peripheral – loosely attached to the outer or inner surface of the lipid bilayer Lipid-anchored proteins – covalently attached to lipid molecules within the bilayer Inner leaflet vs. outer leaflet of lipid bilayer Inner = faces cytoplasm and involves proteins in intracellular signaling Outer = faces extracellular environment, proteins for signaling Vesicle Small membrane 0-bound sac that transports substances Exocytosis Process where vesicles fuse with the plasma membrane to release their contents outside of the cell Membrane fluidity (crystal/solid, gel, fluid) crystal/solid–ridged state at low temps Gel – semi-solid state at intermediate temps Fluid – flexible state at higher temps Lipid packing (length vs. saturation) Length – longer fatty acid chains reduce fluidity Saturation – saturated fatty acids reduce fluidity, unsaturated incidence Cholesterol Lipid molecule that modulates membrane fluidity and stability Anterograde vs. retrograde Anterograde = ER to golgi to plasma Retrograde = backwards Coat proteins, clathrin (triskelion) Coat proteins = proteins that shape vesicles and select cargo for transport Clathrin = forms a triskelion shape, and creates polyhedral lattice for vesicle formation Dynamin GTP enzyme that helps “pinch off” vesicles during endocytosis Actin vs. microtubules Actin = thin filaments for short distances Microtubules = thick filaments for long distances SNARE proteins (T-SNARE and V-SNARE) tSNARE = target membranes that assist in vesicle fusion vSNARE = located on vesicles and interacts with tRNARES to facilitate membrane fusion G-proteins (GAP, GEF) GAP = regulates g-protein activity by promoting GTP hydrolysis GEF = activates g-proteins by promoting the exchange of GDP for GTP Ricin: RIP, lectin, A-chain, B-chain, glycosidase, Sec61 (translocon), endocytosis RIP - inhibits protein synthesis Lectin = binds to carbohydrate A-chain = inactivates ribosome B-chain = facilitates entry into the cell by binding to cell surface carbohydrate Glycosidase = enzyme activity of the A-chain that cleaves glycosidic bonds Sec61 = protein complex that aids in the translocation of polypeptides across ER Endocytosis = process where cells internalize substances from the extracellular environment When things go wrong Predict the result if a cell was unable to degrade misfolded proteins or remove damaged organelles. Accumulation of misfolded proteins and protein aggregation Outline the three main processes that occur in the ER to properly fold proteins and the components that are required for each. Chaperone-mediated folding Molecular chaperones bind to polypeptide chains to prevent premature fielding. Formation of disulfide bonds PDI helps to form disulfide bonds that are formed between cysteine within a protein that stabilizes 3D structure Glycosylation OST - add carbohydrate chain that aids in proper folding and stability Describe three reasons for a misfolded protein (Mistake in transcription or translation/mutation/global problem with cellular conditions) Mistake in transcription or translation Mistakes during transcription of DNA into mRNA lead to incorrect mRNA sequences Errors in translation can cause wrong amino acids Genetic mutation Production of proteins with altered amino sequences Global problems Heat shock, stress, or changes in pH can denature proteins or interfere with foldings Outline the three different fates of misfolded proteins. Refolding Can be refolded to its proper functional state Degradation Targeted for degradation by the proteasome or lysosome Aggregation Can form aggregates accumulating within the cell Describe the purpose of ERAD. In the ER that ensures proper folding and function of proteins Detect misfolded proteins, retrotranslocation, degrade proteins Contrast the conditions that initiate ERAD to those of protein refolding. ? ERAD Proteins have severe or irreplaceable misfolding and accumulated Protein refolding Proteins have mild or temporary misfolding Outlines the four steps of protein degradation via ERAD about location and major components.? Recognition ER lumen, molecular chaperones, misfolded proteins identified Retrotranslocation ER membrane, Sec61, transported from the ER lumen to the cytosol through translocon Ubiquitination cytosol, ubiquitin, misfolded proteins are tagged with ubiquitin molecules that mark them for degradation Degradation Cytosol, proteasome, breaking them down into amino acids Describe how the structure of the proteasome relates to its function.? Protein complex responsible for degrading misfolded, damaged, unneeded proteins 20S - cylindrical structure with 4 stacked rings, degrades proteins into small peptides 19S - cap-like structures that unfold proteins Describe the structure and function of ubiquitin as it relates to ERAD.? Small protein that is compact and contains lysine residue Function Tag misfolded protein degradation via the ERAD pathway Ensure that defective proteins are efficiently removed Draw the path of a misfolded protein from the ER to the proteasome. Recognition in the ER (identified by chaperones) Retrotranslocation (transported from ER lumen to cytosol via Sec61) Ubiquitination (molecules are attached to misfolded protein by the series of enzymes Transport to the proteasome (protein recognized and transported to the proteasome) Degradation by the proteasome Describe the cellular conditions that result in the initiation of the UPR.? Activated in responses to cellular stress that affects protein folding in the ER Accumulation of misfolded proteins, ER stress, high protein synthesis demand, genetic mutations Compare the three major cellular processes that can be modified by the Unfolded Protein Response (UPR)? Protein folding Increases the production of chaperones Protein degradation Upregulates the components of the ERAD pathways to degrade misfolded proteins Protein synthesis Reduces overall protein synthesis reduces the influx of new proteins in the ER Compare and contrast autophagy and proteasome-mediated degradation (including initiation, targets, and location in the cell).? Degrade unwanted proteins, autophagy large structures and proteasomes target specific proteins Autophagy Initiation = initiated by cellular stress etc that forms a double-membrane vesicle that engulfs cytoplasmic material Targets = targets large structures Location = cytoplasm and lysosomes Proteasome-mediated degradation Initiation = marking proteins for degradation Targets = targets misfolded, damaged, unneeded proteins Location = cytoplasm and nucleus Analyze how the dynamic nature of mitochondria and autophagy helps protect a cell from damage. Mitochondrial Fission and fusion Maintain healthy mitochondria by segregating and diluting damages Adapt to changing cellular conditions Autophagy Removes damaged mitochondria and other cellular debris Supports cell survival during stress by recycling components Key Words Unfolded proteins? Proteins that have not achieved their proper 3d structure Protein aggregates? Clusters of misfolded proteins that accumulate in a cell Free cysteines (side group) in proteins? Cysteine amino acids with unpaired thiol groups that can form disulfide bonds Inappropriate glycosylation? Incorrect attachment of sugar molecules to proteins that affect function Exposed hydrophobic domains? Regions of a protein that should be buried within the structure but are exposed due to misfolding Chaperone proteins? Proteins that assist in the proper folding of other proteins and prevent misfolding PDI (Protein disulfide isomerase)? The enzyme that catalyzes the formation and rearrangements of disulfide bonds in proteins Glucosyltransferase The enzyme that adds glucose residues to proteins Sec61? Protein complex that forms a channel in the ER membrane, facilitating protein translocation Ubiquitination? Process of attaching ubiquitin molecules to proteins that mark them for degradation by the proteasome Unfolded protein Response (UPR) Cellular stress response activated by the accumulation of unfolded proteins in the ER BiP? Chaperone protein in the ER that assists in protein folding and is involved in UPR Receptor Proteins? Proteins on the cell surface or within the cell that bind to specific molecules Protein Load? Amount of protein synthesis and folding actively occurring within the ER Autophagy The cellular process of degrading and recycling damaged organelles and proteins ? Basal autophagy? Continuous, low-level autophagy that occurs under normal conditions for cellular maintenance Induced autophagy? Increases autophagy triggered by stress conditions such as nutrient deprivation or damage Lysosome Organelles containing digestive enzymes that break down waste materials ? Apoptosis Programmed cell death, is a controlled process that eliminates damage or unwanted cells ? Hypoxia Condition of low oxygen levels in tissues ERAD? Quality controls mechanism that identifies and degrades misfolded proteins from the ER Proteasome catalytic subunit (20S)? The core component of the proteasome responsible for protein degradation Regulatory subunit (19S)? A part of the proteasome that degrades indeed or damages proteins. Specifically recognizes and unfolds proteins and then feeds them into the core of the proteasome Holoenzyme (26S) Complex proteasome that functions as a machinery to degrade unwanted proteins into smaller peptides