Molecular Biology Exam 3 Review Guide PDF

Davis-1 Molecular Biology Exam Three Review Guide Transcription Factors Diagram and describe promoter elements that regulate gene expression. o The core promoter is the binding site for general transcription factors (transcription factors that will be there no matter wh...

Davis-1 Molecular Biology Exam Three Review Guide Transcription Factors Diagram and describe promoter elements that regulate gene expression. o The core promoter is the binding site for general transcription factors (transcription factors that will be there no matter what, that every gene needs to be active) ▪ The TATA Box- a DNA sequence found in the core promoter region that indicates the point at which a genetic sequence can be found read and decoded. Alternation between adenosine and thymine. The promoter tells RNA Polymerase where to start reading a gene. o Inhibitor Element (INR)- the simplest functional promoter that can direct transcription without a TATA Box. o DPE (Downstream Promoters Elements)- Functions cooperatively with the INR for the binding of TFIID in transcription of core promoters in the absence of the TATA Box. o Core Promoters are about 60 nucleotides. o Regulatory Promoter- Where Specific transcription factors bind transcription factors that are required for that specific gene to be activated. o Enhancer- upstream distant enhancers and repressors more than 1000 between 10,000 base pairs away from transcription start site. DNA is bent back to come in contact. Identify transcription factor consensus binding sites on a strand of DNA. o Transcription factors are proteins that bind to specific DNA sequences. o DNA sequences that transcription bind to are called cis element, regulatory element, or the motif. o The transcription factor has a nuclear localization signal which allows the protein to get into the nucleus and get to the DNA. o Transcription factors have DNA binding domains is where it binds the specific sequence. o Transcription factors have activating/ repressing domains which regulate whether it’s needed or not. Distinguish between types of binding domains. Davis-2 Davis-3  Davis-4 RNA Synthesis Summarize the roles of different eukaryotic RNA polymerases. o RNA Polymerase I makes rRNA. o RNA Polymerase II makes mRNA, miRNA, and lncRNA. o RNA Polymerase III makes tRNA. o RNA Polymerases IV and V make plant specific RNA directed DNA methylation. Describe the function of general transcription factors for RNA polymerase II. o mRNA is made by RNA Polymerase II. o Initiation- where the promoter comes in and defines the start of a gene. o Elongation- get transcription bubble, separates helix so you can copy into RNA, DNA gets back together as RNA polymerases passes through. o Termination- RNA polymerase falls off, DNA is unchanged, mRNA has been created, RNA polymerase is unchanged. Outline the steps required initiation, elongation, and termination. o Initiation ▪ TFIID (Transcription Factor II (Signifies which DNA Polymerase)) Binds to TATA box in section called TATA binding protein (TBP) TBP-associated factors (TAF)- hug protein complex where only one binds to protein the rest are just there. ▪ TFIIB Start to build initiation complex, goes from TFIID to the binding of TFIIB, B binds to TATA box binding region then recruits TFIIF. ▪ TFIIF and RNA Polymerase II F binds to TFIID and TFIIB and brings in polymerase ▪ TFIIE and TFIIH Binds to complex. ▪ Mediators Mediators join (bunch of proteins required for transcription). ▪ Phosphorylation of RNA Polymerase II CTD by TFIIH releases RNA Polymerase II On RNA polymerase there is a beaded tale known as the C- terminal domain (end part on C terminus where the central group is sticking out, part of protein). To remove RNA Polymerase II the C- Terminal Domain is phosphorylated by kinase domain found of TFIIH. Without phosphorylation means RNA polymerase just sits in the pre-initiation complex. Mutations that result in the loss of kinase function for TFIIH are lethal, because TFIIH loses its ability to phosphorylate no RNA can be made and no proteins. o Elongation ▪ RNA Polymerase II 12 Subunits  Davis-5 ▪ Histones are in the way, they keep RNA polymerase from reading, the helix needs to be split and that cannot be done if bound to proteins. ▪ FACT Facilitated Chromatin Transcription Protein Breaks nucleosomes as RNA polymerases are coming through and put them back together as RNA polymerase passes by Opens gate allows RNA polymerase to go. ▪ Transcription Bubble The active site of RNA Polymerase Iihas a channel that splits DNA, as DNA comes in one strand is forced up into the channel, the other strand goes into the secondary channel allowing for the RNA to complement. Only about 25 base pairs are separated once it reaches the other DNA reforms. 8 base pairs at a time DNA/RNA hybrid. RNA is passed out exit channel and keeps going. o Termination ▪ Pre-mRNA is cut off, but RNA polymerase keeps going. At polyadenylation signal where poly A tail is added mRNA is cut and released. ▪ 5’ exonuclease (XRN2 for humans) Starts eating up the RNA until it catches up to the polymerase and bumps it off. Gets rid of RNS that shouldn’t be in the cell and ends transcription. Compare and contrast the mechanisms of initiation, elongation, and termination. o RNA Polymerase I ▪ Initiation Upstream binding factor I (UBFI) Selectivity Factor 1 They bind to promoter and polymerase I is recruited and released. Protein I bind to promoter, another promoter binds to protein, polymerase is brought to promoter, and polymerase is released. ▪ Elongation Same as RNA Polymerase II ▪ Termination TTF-1 o Transcription Termination Factor for RNA Polymerase I o RNA Polymerase III ▪ Initiation TFIIA TFIIC  Davis-6 TFIIB Polymerase III ▪ Elongation Same as RNA Polymerase II ▪ Termination A stretch of 4-7 Us triggers disassociation (As on DNA). When basically the whole transcription bubble is full of Us.  Davis-7 RNA Processing Summarize the event involving processing rRNA. o 5.8s, 18s, and 28s derived from a single transcript. The transcript is cut up to make all three. 5s is made from a separate transcript. o Pseudouradine is formed through isomerization of ribose uradine. o Methylation- a methyl group is added to the OH group. o Organization required: ▪ snoRNPs (small nucleolar RNA proteins) 8-10 proteins Bind together the different pieces of rRNA. 2 families Small nucleolar RNA (snoRNA) ▪ Need structure and scaffolding before being placed in the ribosome because structure determines function, proteins make sure base pairs are in the right spot, right orientation, and prevent base pairing with lose RNA. Summarize the event involving processing tRNA. o Cytoplasmic tRNA scattered throughout genome (lots, everywhere). o Transcript comes out as line so must be processed into proper shape. o Catalyzed by RNase P ▪ RNA enzyme, no protein, riboenzyme ▪ Cleaves ▪ Extra base pairs on both sides, molecular scissors o CCA addition- needed to bind a peptide and the codon, on 3’, allows to stick out enough so amino acid can bind and is far enough away. o Base Modifications are required at some spots. Summarize the event involving processing mRNA. o 5’ Capping ▪ 5’ end gets capped at the first piece of RNA to exit RNA Polymerase II, to keep RNA polymerase from degrading, occurs after immediately leaving polymerase. ▪ 7-methylguanosine (based on guanine) creates linkage between 5’ ends of two bases 5’-5’ linkage ▪ Nothing can bind or attach, it is protected, serves as marker for exit/ ▪ Capping enzymes associated with RNA Polymerase II Negative fac tors NELF and DSIF bind and pause elongation. o Occurs during elongation because 5’ end needs to come out of RNA polymerase. CE adds cap to RNA. P-TEFb (kinase) phosphorylates NELF, and DSIF (the negative factors that pause elongation). Phosphorylation changes the protein  Davis-8 shape of these proteins causing them to fall off the polymerase resuming elongation. Elongation resumes o 3’ Polyadenylation ▪ Polyadenylation signal- the signal that signals the transcript is done. Hexanucleotide Sequence AAUAAA within transcript Upstream Element Downstream Element o U or GU rich The upstream and downstream elements allow enzymes to know where to bind and which stretch of AAUAAA is the poly A signal. ▪ Endonuclease- snips after protein binds. ▪ Poly-A-polymerase- puts lots of As together 200 (PAP). o Splicing ▪ The spliceosome is a big collection of enzymes that recognize the starting point, the branch point, and the end point. 5’ splice site Branch Point 3’ Splice Site Controlled by a Spliceosome. Compare RNA production and processing.  Davis-9 RNA Processing Two Outline the assembly of the spliceosome. o Smal nuclear RNA (snRNA) ▪ U1 ▪ U2 ▪ U4 ▪ U5 ▪ U6 o All come together to make a small nuclear ribonucleoprotein (snRNP) ▪ snRNA + Proteins o These will all come together to make up the spliceosome (a great complex that associates with polymerase II the spliceosome as RNA leaves polymerase II recognizes introns and splice them during elongation as its coming out of RNA polymerase II and you end up with two spliceosomes they are working together to cut the intron and put the exons together now we have two exons and a little bit of RNA that will float away. Describe the roles of snRNAs splicing. o U1 ▪ Binds to 5’ splice site, the part that leaves RNA polymerase, the first thing to bind. Marks intron. o U2 ▪ Bind to branch point, A, within the intron. o U4/U6/U5 ▪ A complex that is bound together to bring in U6. ▪ U5 binds to U2. Complex hanging on building loops. ▪ U5 is bound to U2 and U6 keeping everything where it needs to be. o U1 & U4 Dissociate ▪ Remove U1 because bound to splice site (blocking). ▪ Remove U4 because its job is just to bring U6. o U6 ▪ Binds to 5’ splice site. ▪ Catalyzers splicing then ligation. o U1 binds to one side of the intron and U2 binds to the other and brings U1 and U2 together. Then U4/U6/U5 complex comes in and binds where U5 binds to U2. U1 and U4 leave. The U6 cuts both sides and ligases each side removing the introing and reconnecting the mRNA.  Davis-10 Epigenetics Describe the effect of different histone modifications on transcription. o Epigenetics controls whether an area is tightly packed heterochromatin or loosely packed euchromatin. o Acetylation ▪ Associated with transcription. ▪ Histone Acetyltransferase Adds acetyl group to a protein, always a lysine. ▪ Histone Deacetylases Remove acetyl groups. Active Sites ▪ Determines whether high density and low transcription or low density and high transcription. ▪ Determines whether an area is allowed to have RNA polymerase show up. o Methylation ▪ Non active site and Active Site. o Phosphorylation ▪ Active Site o When adding acetyl group, methyl, or phosphate changes the function, less tightly because bound to other groups, changes shapes of tail. Summarize the action of chromatin remodeling factors. o Large family of proteins o Require ATP o Mechanisms ▪ Slide histones apart, exposing, the stretch of DNA that has the promoter where the initiation complex built. ▪ Also, can be inhibitors. ▪ Inducing structural changes ▪ Remove histones from DNA. ▪ Makes so other proteins. Describe the action of lncRNAs in gene repression and activation. o Scaffolds o Recruits’ chromatin remodeling factors o Ensures transcription area is accessible. o Loosen and tightened back out. o One side is opening the field, the other is tightening it back up.  Davis-11 Quiz 5 1. Transcription is ____ dependent ______ synthesis. a. DNA; DNA b. RNA; DNA c. DNA; RNA d. RNA; Protein 2. Eukaryotic RNA polymerase I genes code for a. Small nuclear RNAs and small cytoplasmic RNAs b. mRNAs c. tRNAs d. rRNAs 3. The first step in the formation of a transcription complex for mRNA transcription is the binding of _____ to the TATA box. a. TFIIA b. TFIIB c. TFIIE d. TFIID 4. Eukaryote mRNA processing occurs in a. The Golgi apparatus b. The nucleus after completion of transcription c. A complex with RNA polymerase d. The cytoplasm 5. The RNA components of the spliceosome are five different. a. siRNAs b. small nuclear RNAs c. micro RNAs d. small cytoplasmic RNA 6. Eukaryotic gene repressor proteins are thought to act by binding to a. Specific activating proteins, preventing their binding to DNA. b. Basal transcription factors, inhibiting transcription c. All of these are correct. 7. Identification of transcription factor DNA binding site can be most accurately measured using… a. PCR b. Electrophoretic Mobility Shift Assay c. Yeast-2-Hybrid Assay d. Bioinfographics 8. How do long noncoding RNAs (lncRNAs) function in regulating gene expression? a. They form scaffolds to help stabilize protein complexes that modify chromatin. b. They bind to complementary DNA sites and interfere with RNA polymerase binding. c. They assist with DNA loop formation between enhancers to facilitate transcription initiation. d. They catalyze the modification of histones to stimulate chromatin condensation. 9. Chromatin remodeling factors are responsible for shuffling and rearranging DNA sequences in chromatin to make them either more or less accessible for transcription. a. True b. False  Davis-12 10. Which statement about cis-acting elements is true? a. They are specific DNA sequences that control transcription of adjacent genes. b. Various specifically recognize and bind to these cis-acting sequences. c. All of these are correct. d. They may be directly adjacent to the gene they control or far away.  Davis-13 Translation Pt 1 Describe how tRNA are activated for use in translation. o Anti codon loop interacts with mRNA. o CCA amino acid attachment site- where amino acids attach, requires enzyme aminoacyl tRNA synthetase, this family of enzymes is responsible for tRNA and attaching it to the correct amino acid, every time it does that it requires energy takes ATP →AMP (Tri→Mono) breaks two high energy phosphate bonds to have enough energy to attach an amino acid to the tRNA. o tRNAs must be activated. ▪ aminoacyl tRNA synthetase ▪ Requires energy. o Aminoacyl – tRNA ready for use o Cell keeps set amount of tRNA ready to go for at any point you need to start translating something happening constantly, as soon as used tRNA is reactivated and amino acid is put back on o Every amino acid has its own synthase. Describe the structure and function of ribosomes. o Eukaryotic ribosome 80s ▪ 60s large subunit 28s, 5.8s, 5s rRNA +34 proteins Catalyze RNA forms peptide bonds where peptide bonds are created as amino acids come in from one tRNA. It is in the 60s large subunit they are put together. ▪ 40s small subunit 18s rRNA +33 proteins o When the ribosome gets together it forms three sites, A, P, and E Sites ▪ Part of the sites are both the large and small subunits. ▪ A- charged amino acyl- tRNA binds. ▪ P- Where the peptides are bound together. ▪ E- exit, non-charged tRNA exits. ▪ As tRNA comes in and the ribosome shifts your tRNA moves to the next site ▪ When the ribosome is called for protein production the small subunit finds the mRNA in the cell and after they get together, they join together with the larger subunit. Hamburger, large subunit is the large top unit, the meat is the mRNA, small ribosomal subunit is the small bottom bun, breaks apart after completing the job.  Davis-14 Outline the events of translation initiation. 1. eIF3 (eukaryotic initiation factor) keeps ribosome subunits from coming together until its time, bounds to the small subunit and makes sure the large subunit stays away until its time. 2. eIF2 + GTP brings in the original met charged tRNA. 3. eIF4 complex is binding the mRNA, you have 40s subunit that has a met, responsible for binding to mRNA. 4. mRNA + 40s + met-tRNA held together by eIFs, brought together, eIF4e bound to 7 guanosine caps, brings in the mRNA and lines it up with the ribosome subunit and this is all held together by a bunch of eIFs. 5. Caps scans mRNA for AUG (recognized by met-tRNA, anticodon UAC) the complex attached at the cap so must scan for start codon. 6. eIFs dissociate from small subunit, eIF5B recruits large subunit. 7. Now the ribosome is fully formed with met-tRNA in P-site, all initiation factors went away. o GTP was released when the 60s came in, ATP used for scanning, 1 GTP to bring in a charged tRNA molecule, 3 ATP energy molecules. Outline the events of translation elongation. 1. eEF1α (eukaryotic elongation factor) brings in the next charged tRNA, costs energy. 2. Inside the 40s is the decoding center and takes place in the A site.  Davis-15 a. If tRNA binds quickly GTP is used to dissociate the elongation factor away. If tRNA anticodon matches GTP→GDP occurs. b. If not matched right the tRNA diffuses out because it does not bind. If tRNA anticodon doesn’t match, tRNA dissociates before GTP→GDP occurs. c. Proofread so the wrong amino acid does not bind. 3. eEFLα dissociates. 4. Riboenzyme forms peptide between amino acid in P site and amino acid in A site, inside ribosome. 5. Ribosome Translocation a. eEF2-GTP→eEF2-GDP, causes break between original tRNA and amino acid and the ribosome moves. b. Ribosomes moves 3 nucleotides=1codon. c. Requires energy. 6. tRNA shifts to the next site. a. tRNA in A→P b. tRNA in P→E 7. The cycle repeats until it finds the end. 1 ATP to change tRNA, 1 ATP to move ribosome, 2 ATP per amino acid. Outline the events of translation termination. o Stop Codons ▪ UAA, UAG, and UGA ▪ No tRNA with complementary anticodons ▪ Ribosome just hangs out till release factors bind to stop codon, that releases final tRNA peptide bond, stop codon binds and cuts the polypeptide, everything falls apart. Evaluate the cost of translation per amino acid. o 1 ATP to change tRNA, 1 ATP to move ribosome, 2 ATP per amino acid.  Davis-16 Translation Pt 2 Outline the events of translation initiation. Outline the events of translation elongation. Outline the events of translation termination.  Davis-17 Explain translation in organelles. o Mitochondria and chloroplast have their own translation machinery. o The ribosomes in organelles resemble that of prokaryotes. ▪ 70s (50s + 30s) ▪ Ribosomes are prokaryotic not eukaryotic, so it is a different size. o Polysomes ▪ Multiple mRNAs ▪ Because everything is just hanging out in the organelle and there is no nucleus you have a bunch of ribosomes translating the mRNA as it is being made there is no moving mRNA out to the cytosol, so you get simultaneous transcription and translation occurring at the same time.  Davis-18 Translation Pt 3 Describe the methods of regulating translation. o Translation Regulation via mRNA regions ▪ Regulatory proteins bind to 5’ UTR mRNA and thus prevent the 40s (ribosome) from binding to the mRNA. Ribosome Side ▪ Translational repressor bound to 3’ UTR of mRNA preventing eIF4 complex formation. Blocks translation eIF4 brings in the mRNA it is bound to that 3’ UTR of mRNA, but if there is a regulatory molecule bound to the 3’ UTR then eIF4 can not bind there so the mRNA can not be taken to the translation party. Initiation Factor side ▪ miRNA bound to mRNA prevents ribosome translocation. miRNA sites in open reading frame and say no you can’t pass. Blocks during elongation. Blocks specific gene (gene specific). ▪ siRNA also would bind in a protein coding region of mRNA but would cut up mRNA. Regulates translation indirectly. o Translation regulation via disruption of initiation ▪ Cell wide disruption of translation ▪ Messing with initiation factor, the proteins themselves, instead of interacting with mRNA we are going to impact the initiation factors themselves. ▪ Targets eIF2-brings in0 met-tRNA. o Phosphorylation of eIF2 + GDP prevented. o Starvation of eIF2 + GTP ▪ Not enough energy to carry out function, can’t regenerate GTP (blocked) o Can be triggered by a protein that phosphorylates the initiation factor, adding a phosphate group makes it so it can no longer hold onto the GDP. ▪ Sometimes this happens because there is not enough energy to replace the GDP, sometimes the cell targets this to temporarily block translation (reversible, another protein to remove phosphate) eIF4E-binds to cap and binds all other stuff. o Protein, 4E-BP (eIF4E binding proteins), block synthesis pf eIF4 complex.  Davis-19 ▪ Protein-protein interactions ▪ 4E-BP in the way of from others binding. o Kinases phosphorylate 4E-BP for translation to occur. ▪ Phosphate blocked from finding to eIF4E.  Davis-20 Post-Translational Processing Describe the function of enzymes that facilitate protein bond formation. o Protein’s 3D structure is imperative for function. o Protein folding is driven by interactions between amino acids. ▪ Interactions between R groups determine shape and the chemical properties of the R groups. o Enzymes can be involved in protein folding. ▪ Protein disulfide isomerase Catalyze disulfide bonds, makes disulfide bonds, a sulfur on one R group attaches to a sulfur on another R group. ▪ Peptidyl Propyl Isomerase Isomerization proline from cis to trans, takes R group from one side of central carbon to the other. H-C-R→R-C-H Describe the mechanism of chaperones and chaperonins. o Chaperones ▪ Large Group of helpers ▪ Proteins that fold other proteins ▪ The largest group is heat shock proteins. ▪ Chaperones bind to the linear polypeptide chain as it is coming out of the ribosome and then after everything is out of the ribosome helps facilitate that folding into the final 3D structure. ▪ There are many chaperone systems that are basically two heat shock proteins that work in tandem and that’s how we name them. Heat shock protein 70 is the combination of heat shock protein 70 and 40. Names based on which two heat shock proteins are interacting with each other. ▪ Found in the cytosol, ER, mitochondria, and chloroplast. ▪ HSP4- delivers protein to HSP70. HSP70 has a nucleotide related factor so it has an ATP associated with it and it’s going to need ATP to fold the protein. ▪ ATP hydrolysis (ATP→ADP) causes HSP70 conformational change. ▪ Peptide forms ▪ NEF swaps ADP for ATP in HSP70, another conformational change, open. When it has ATP its Open, when open the folded protein is released. When it has ADP, it is closed, and the protein is folded. ▪ The folded protein is released, HSP70 is reset. o Chaperonins ▪ A type of chaperone All chaperonins are chaperones but not all chaperones are chaperonins, ▪ GroEl/GroES in bacteria, HSP60/10 in Eukaryotes  Davis-21 ▪ Denatured protein is loaded into HSP60(GroEL) ▪ HSP10 uses ATP and attaches to top creating a closed system, a closed system can have its hydrophobic/hydrophilic ratios change because it is no longer in the cytosol. ▪ ATP hydrolysis induces conformation change which provides correct microenvironment for protein folding. Provides the correct environment, closes, and bring s forth a hydrophobic amino acid that was previously in the barrel now its sticking out and can interact with other hydrophobic in the region so the conformation change will change what the microenvironment that the protein is given. Changing the microenvironment provides the perfect protein folding environment for protein. ▪ ADP dissociates which causes HSP10(GroES) to dissociate allowing for the release of the newly folded protein. ▪ When a cell is under heat stress proteins unfold. Differentiate between chaperones and chaperonins. o Chaperones and chaperonins provide a way for denatured proteins to be refolded.  Davis-22 o Chaperonins are typically ribosome related and chaperonins are typically environment related. o Chaperones refer to the proteins which assist the covalent folding or unfolding and assembly and disassembly of other macromolecular structures while chaperonins refer to the proteins which provide favorable conditions for the correct folding of denatured proteins, preventing aggregation. Thus, this is the fundamental difference between chaperones and chaperonins. o Most of the chaperones are heat shock proteins (HSPs) while chaperonins have the shape of two donuts stacked on top of one another to create a barrel. o Furthermore, another difference between chaperones and chaperonins is that the chaperones are responsible for the folding, unfolding, assembly, and disassembly of proteins, while the chaperonins are responsible for the correct folding of denatured proteins, which prevent aggregation. Explain how glycosylation and lipidation of proteins. o Glycosylation – addition of sugars ▪ Addition of a carbohydrate to a protein Glycoproteins=protein with some sugar ▪ N Linked The sugar is added to a nitrogen in asparagine. ▪ O Linked The sugar is added to an oxygen in serine or threonine. o Lipidation- addition of fats (lipids). ▪ The addition of lipids to proteins. Can also add lipid to a carb. ▪ Often in membrane related proteins because lipids like lipids. ▪ Glycolipid protein = carbohydrate + lipid on a protein.  Davis-23 Explain how proteolysis can convert an inactive precursor to an active protein. o Cleavage of polypeptide chain o Usually has two purposes: ▪ Get rid of a signal peptide which sends protein to correct location (signal sequence = zip code). ▪ Cleaving to activate a protein, get rid of protein that keeps it inactive (inactive→ active).  Davis-24 Quiz 6 1. Aminoacyl tRNA synthetases are enzymes that... a. connect amino acids while they are held in place on ribosomes by transfer RNAs. b. attach amino acids to specific transfer RNAs. c. synthesize transfer RNAs. d. attach the terminal CCA sequence to transfer RNAs. 2. Translation always occurs on which of the following structures? a. Nuclear envelope b. Endoplasmic reticulum c. Ribosomes d. Mitochondria 3. The first step in the initiation of protein synthesis is the binding of _______ to the _______. a. initiation factors; small ribosomal subunit b. the initiator tRNA; initiation codon c. initiation factors; initiation codon d. the small ribosomal subunit; initiation codon 4. The factor that escorts the aminoacyl tRNA to the eukaryotic ribosome and then releases it with GTP hydrolysis following the correct codon–anticodon base pairing is... a. eIE2 b. eRF1 c. eEF1a d. eIF1 5. Termination of translation and release of the polypeptide chain occur when… a. a protein release factor binds to the termination codon. b. a tRNA with a complementary anticodon binds to a termination codon, and a release factor bound to the CCA end releases the chain. c. a small molecule shaped like puromycin binds to the termination codon. d. tRNA binds to a termination codon via a complementary anticodon but lacks an amino acid. 6. The correctly folded three-dimensional configuration of a protein is determined primarily by the… a. pathway by which it folds. b. chaperones with which it interacts. c. sequence of nucleotides of its gene. d. primary sequence of its amino acids. 7. Many chaperones are called heat-shock proteins because they... a. denature at high temperatures. b. misfold during a heat shock. c. cause fever in mammals. d. are expressed at higher levels after a heat shock than under normal growth conditions.  Davis-25 8. In a major protein degradation pathway, a short polypeptide called _______ is attached to a protein to target it for destruction. a. ubiquinone b. KDEL c. glutathione d. ubiquitin 9. The half-life of proteins in the cell varies widely, ranging from... a. milliseconds to seconds. b. minutes to days. c. days to weeks. d. three to seven minutes. 10. Proteins are often regulated by phosphorylation, which is catalyzed by enzymes called... a. protein phosphorylases. b. phosphoproteases. c. protein kinases. d. protein phosphatases.  Davis-26 Protein Regulation Describe cellular signaling. o Cellular signaling is a pathway that translates an external signal, a signal from outside the cell, into some kind of cellular function. Explain how protein-protein interactions play regulatory roles. o Regulatory-catalytic subunits o Dimerization/ Trimerization- two or three of the same proteins. o Coming together of two membrane proteins around a signal molecule and its not till they come together that they are active. o Makes sure things don’t turn on when they are not supposed to turn on, make sure you are not wasting energy if you don’t need to be. Identify the small molecules used involved in post-translational regulation. Davis-27  Davis-28 Explain how the binding of small molecules can regulate protein function. o Binding targets conformational change o Active →Inactive  Davis-29 Protein Degradation Outline the process of polyubiquitination. o Translation builds protein. o Degradation destroys proteins. o Half-lives of proteins ▪ Minutes→Days o 78 amino acids, ~KDA o Ubiquitin ▪ Polyubiquitination=degradation ▪ Ubiquitination=signaling o Ubiquitin attached to Ubiquitin-activating enzyme (E1) ▪ Requires ATP ▪ 2 in humans o Ubiquitin transferred to ubiquitin-conjugating enzyme (E2) ▪ 38 in humans o Target protein bound to ubiquitin ligase (E3) ▪ >600 in humans o E3 and E2 bind. o Ub transferred from E2 to target protein. o Polyubiquitinated proteins recognized by proteosome. o Proteosome binds poly-Ub-signal. o Protein broken down in proteosome. ▪ Requires ATP o Peptides released. o Peptides can be used to synthesize new proteins or further degraded.  Davis-30 Describe the structure of the proteasome and its function. o 26s ▪ Two subunits ▪ 19s Regulatory PolyUb binds 19s lid. Protein unfolded and fed into catalytic subunit. o Requires ATP 19s base exit channel. ▪ 20s Catalytic subunit 4 Rings 6 Protease Sites Cleavage results in 2-25 amino acid polypeptides. o Cytoplasmic protease ▪ Degrades proteins in cytoplasm, o Nuclear Protease ▪ Degrades transcription factors in nucleus.  Davis-31 Describe the UPR and the consequences of its failure. o ER stress due to accumulation of misfolded/unfolded proteins. o Unfolded protein response. ▪ Lowers global protein production. ▪ Upregulates membrane lipid biosynthesis. ▪ Induces ERAD (endoplasmic-associated protein degradation) pathway (E3 ligases) ▪ Expands Secretory Pathway (Er-Golgi) ▪ Chronic UPR→Apoptosis  Davis-32 Quiz 5 Key 1. C 2. D 3. D 4. C 5. B 6. D 7. B 8. A 9. A 10. A  Davis-33 Quiz 6 Key 1. B 2. C 3. A 4. C 5. A 6. D 7. D 8. D 9. B 10. C