Unit 4 Vocab and Questions
Unit 4 Vocab and Questions BIO 204
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Replication laying down new nucleotides causing an incorrect pairing The process through which complimentary to the template that could lead to point the genetic information of a strand on both the leading and mutations due to DNA’s lagging strand after the RNA semiconservative replication cell is copied through a series of enzymatic reactions to pass primer has been established properties identical DNA to daughter o alphasubunit: Enzymatic Telomere cells. polymerization of DNA in Repetitive DNA added to the 5’ to 3’ Polarity 3’ ends of chromosomes early the 5’ 3’ direction refers to the carbon numbers o epsilonsubunit: in development of the sugars ribose and Exonuclease that proofreads End replication problem deoxyribose which contain in a 3’5’ direction occurs because lagging hydroxyl groups to which strands would be staggered o betasubunit: holds the leaving them uncopied incoming nucleotides are whole complex onto the attached during DNA strand Telomeres prevent the polymerization, with addition o tausubunit: Tethers the two staggered ends from specifically to the 3' hydroxyl alpha subunits of DNA activating the cells systems group, resulting in increases for monitoring damaged in length at the 3' end, in polymerase III to each other DNA. DNA Polymerase I polymerization in the 5' to 3' Replaces the RNA primer Also acts as a buffer that direction with DNA after RNase provides protection against Antiparallel the organism’s genes The configuration of a DNA degrades the RNA primer. shortening. They do get Polymerizes in the 5’3’ molecule that is made up of direction shorter after every round of two strands held together by DNA Ligase replication eventually leading complimentary base pairing. to somatic cell apoptosis or This results in one strand An enzyme that connects the senescence= no more cell reading 5’3’ one direction fragments of lagging strand division. DNA and 5’3’ in the opposite Okazaki Fragment Telomerase direction. The enzyme that adds RNA Primase Lagging strand segments that telomeres to DNA strands. An enzyme that lays down contain the RNA primer and Operon nucleotides laid down by complimentary RNA DNA Polymerase III Set of genes that are related nucleotides to the template Leading Strand and coordinate functionally strand of DNA known as for a protein RNA primers. DNA synthesized Includes the switch segment Necessary before DNA continuously 5’3’ following the replication fork (helicase) of DNA called an operator, Polymerase III can lay down Lagging Strand the promoter sequence and the new DNA genes they control. Helicase DNA synthesized in segments Polycistronic The enzyme that “unzips” the away from the replication fork also in the 5’3’ direction Describing a type of original DNA strand to start Exonuclease messenger RNA that can replication and create encode more than one replication forks and two An enzyme that works by polypeptide separately within template strands cleaving nucleotides one at a the same RNA molecule. Single strand binding protein time from the end (exo) of a polynucleotide chain. Monocistronic These proteins bind to the Endonuclease An RNA molecule that unpaired, separated DNA encodes for only one protein strands and keep them from An enzyme that works by Promoter repairing. cleaving nucleotides one at a time from the The DNA sequence where Topoisomerase Tautomers RNA polymerase attaches and The enzyme that stays ahead initiates transcription. of the replication fork Rare forms of nucleotides that RNA Polymerase relieving the strain on the yet can “confuse” DNA to be separated DNA because polymerase when the enzyme An enzyme that pries the two selects nucleotides to pair up strands of DNA apart and the untwisting of the double joins together RNA helix causes strain. Nucleotides have a floating nucleotides complementary to DNA Polymerase III proton that can give the the DNA template strand. The enzyme that begins molecule a different structure Assemble a polynucleotide in the 5’3’ direction. for translation. Small nuclear RNA that, TFIID/TBP Poly A Tail together with proteins, make up spliceosomes TATA binding protein that At the 3’ end, Poly A recognizes the promoter polymerase adds about 200 miRNA sequence and recruits general more adenine nucleotide Micro RNA that regulates the transcription factors to bind Used to facilitate the export of expression of genes by and assemble the mRNA from the nucleus, interacting directly with DNA TFIIH used to protect the mRNA or w mRNA copy A helicase enzyme that from degradation by Capable of binding to unwinds parent DNA and exonucleases, and they help complementary sequences in phosphorylates RNA ribosomes identify and attach mRNA molecules. to the 5’ end of the mRNA. polymerase II at the C Allows complex to bind to terminal domain 5’ Cap any mRNA molecule with at Phosphates become binding A modified form of guanine least 7 or 8 nucleotides of sites for “hitchhiker” proteins nucleotide added to the 5’ end complementary sequence. such as Capping Enzymes and after transcription miRNA and protein complex Cleavage factors Done for same reasons as either degrades the target Also used as a promoter PolyA tail. (Export of mRNA or blocks its clearance nucleus, prevent degradation, translation TATA Box point of attachment for Ribozyme A nucleotide sequence ribosomes) RNA molecules that function containing TATA, about 25 Exon as enzymes nucleotides upstream from the Regions of mRNA that are found in the ribosome where transcriptional start point. expressed by being translated they join amino acids together Template Strand into AAs. to form protein chains The strand of DNA that is Intron 5’/3’ UTRs used to make a new strand of Regions of mRNA that are Parts of the mRNA that will RNA during transcription by noncoding segments that are not be translated into protein, matching complimentary usually between coding but they have other functions, nucleotides to it. regions and are cut out by such as ribosome binding. Nontemplate Strand large complex of proteins and Start codon The other strand of DNA not snRNA called splicesomes. Codon AUG that signals the being used to create RNA snRNP proteinsynthesizing during transcription A complex of snRNA and machinery to begin translating Cterminal Domain proteins that bind to introns to the mRNA at that location. The carboxyl end of an amino splice them out of mRNA Stop codon acid where more amino acids Spliceosome Termination codon that are attached to create the The formal name of the signals the end of a growing polypeptide chain complex made up of snRNA translation. during translation and binding proteins that Aminoacyl tRNA Synthetase General Transcription Factor cleave out introns from (AARS) class of protein transcription mRNA Correct matching up of tRNA factors that bind to specific mRNA and amino acid is carrier out sites (promoter) on DNA to Molecule of nucleotides that by these enzymes. activate transcription of is transcribed from DNA and Active site of each AARS fits genetic information from translated into AAs via only a specific combo of AA DNA to mRNA. ribosomes. and tRNA making there be 20 Poly A Signal tRNA different synthases for each Polyadenylation signal Transfer RNA used to transfer AA. sequence in DNA (TTATTT) amino acids from the Catalyzes the covalent that specifies a signal cytoplasmic pool to a growing attachment of the AA to sequence in mRNA polypeptide in a ribosome. tRNA driven by hydrolysis of (AAUAAA). rRNA ATP Once this stretch of RNA molecules that, together Ribosomal Subunit nucleotides appears, it is with proteins, make up Small subunit immediately bound by ribosomes; the most abundant o Binds to both mRNA and a cleavage factors to release the type of RNA mRNA to head to ribosomes specific initiator tRNA, snRNA which carriers the AA polymerase to the promoter Activate or inhibit gene methionine. Binds the preventing transcription of the expression by recruiting mRNA at a specific RNA genes. chromatin modifying enzymes sequence, just upstream Corepressor to genes. from the start codon, AUG Small molecule that Combinatorial Code o Binds to the 5’ cap of the cooperates with a repressor Only a dozen or so nucleotide mRNA and then moves, or protein to switch an operon sequences appear in control scans, downstream along off elements for different genes. the mRNA until it reaches Inducer Each enhancer is composed of the start codon Small molecule that about ten control elements Large subunit inactivates the repressor if the each of which can bind only o Completes the initiation one or two specific tx factors. operon is always active and complex. requires a repressor to turn it The particular combination of o Hydrolysis of GTP off. control elements in an provides the energy for the Catabolite Activator Protein enhancer associated with a assembly. Initiator tRNA agene, rather than the is in the P site; the A site is (CAP) A protein (activator) that presence of a single unique available to the tRNA with binds to DNA and stimulates control element that is the next AA transcription of a gene. important in regulating transcription. “senses” the transport of Histone Acetyltransferase (HAT) another sugar and binds to site Initiation Factors near promoter and interacts Modify chromatin so its more Proteins that are required to with RNA polymerase, open and RNA bring all the translation stabilizes it on a weak polymerase/transcription factors can bind easier and components together. promoter. Elongation Factors o Promoters have specific transcribe a gene. Several proteins that are sequences of nucleotides Histone Deacetylase (HDAC) required to add AAs one by and if sequence doesn’t Remove functional group match ideal sequence of from histone tail causing a one to the previous amino acid at the Cterminus of the promoter, it will not recruit highly condensed chromatin growing polypeptide chain. RNA polymerase well state. lac operon cAMP Methyltransferase A protein that accumulates histonemodifying enzymes Set of genes that are related and coordinate functionally when glucose is scarce that that catalyze the transfer of for lactose allosterically regulates by methyl groups to histone o Lac Z: encodes beta binding to CAP protein. proteins Enhancer gene is brought into a closed galactosidase which cleaves and begin Distal control element which chromatin state so it may not metabolism of lactose maybe thousands of be expressed such as silencing o Lac Y: encodes lactose nucleotides upstream or pancreatic cell from downstream of a gene or even expressing the sequence for permease which is a hemoglobin production. membrane transport within an intron. protein Activator proteins bind to the Heterochromatin o Lac A: encodes enhancer area which has three also referred to as closed galactosidase binding sites. DNA bends via chromatin or gene poor bending proteins that bring because it is inaccessible to transacetylase which is still not understood activators closer to the the machinery of the cell Operator promoter. General responsible for transcribing Positioned within the transcription factors, mediator the genetic information coded proteins, and RNA in the DNA promoter, or between polymerase II are nearby. promoter and enzymecoding Euchromatin genes. Activators bind to certain true chromatin is loosely Controls the access of RNA mediator proteins and general packed making its DNA tx factors helping them form accessible to machinery so the polymerase to the genes. an active transcription genes present in euchromatin Repressor A protein that binds to the initiation complex on the can be transcribed. operator and blocks promoter. Maternal Effect Gene Regulatory Transcription Factor these genes specify polarity of attachment of RNA a developing cell into anterior, tx factors with conserved nurse cell to oocyte and posterior, dorsal, and ventral. DNA binding domains localized to opposite end to Expressed by mother during (homeodomains) become posterior of organism oogenesis (egg development) Similar sequence amongst Bicoid and Nanos mRNA are many organism and they localized at opposite poles of control pattern formation in the oocyte. the late embryo, larva, and adult Segmentation Gene Established by the maternal Bicoid effect gene Gene that is expressed and Determine the position and mRNA is transported from nurse cell to oocyte and polarity of each segment of a developing organism created specifically localized to the by morphogen concentrations region in contact with the that encode for transcription nurse cells to become the factors anterior of the organism Homeotic (HOX) Gene Nanos Homeobox genes encode for Gene that is expressed and mRNA is transported from 1. Where does the energy for the synthesis of nucleic acid molecules come from? How does this necessarily constrain the direction of synthesis of new nucleic acid strands? The linkage of nucleic acid molecules is a dehydration synthesis reaction. Adjacent nucleotides are joined by a phosphodiester linkage consisting of a phosphate group that links the sugars of two nucleotides. The bond results in a repeating patter of sugarphosphate units called the backbone. The two free ends of the polymer are different. One has a phosphate attached to a 5’ carbon and tother end has a hydroxyl group on the 3’ carbon. This restricts a nucleic acid polymer to only be able to add more sugarphosphate molecules to the 3’ end of the previous sugarphosphate structure because during hydrolysis, a water molecule is added and the Oxygen molecule leaves the hydroxyl on the 3’ carbon the oxygen on the phosphate group can bind to the carbon 2. How did the MeselsenStahl experiment provide support for the semiconservative model of DNA replication? How would the results have been different if the mode of DNA replication were conservative? Their experiment eliminated the conservative model for DNA because the first replication produced a band of hybrid DNA. The second replication produced both light and hybrid DNA, a result that refuted the dispersive model and supported the semiconservative model. This proves that unlike a conservative model, daughter cells will contain one old strand from the parent molecule and a newly made strand. A conservative model would have the two parental strands somehow come back together after the process and be seen in its entirety in the daughter cells. 3. What are the specific functions of E. coli DNA Polymerases I and III in DNA Replication? DNA Polymerase III adds DNA nucleotides to an RNA primer using the parental strand as a template to add complimentary nucleotides. DNA Polymerase I replaces the RNA nucleotides of the primer after RNase degrades the primer. 4. Define the functions of the alpha, epsilon, beta and tau subunits of E. coli DNA Polymerase III during replication. Alpha: the enzymatic polymerization of DNA in the 5’3’ direction Epsilon: exonuclease 3’5’ that acts as a proofreader to ensure nucleotides are complimentarily paired Beta: The subunit that clamps polymerase to the DNA strands Tau: Tethers two parental strands and the polymerase to one another. 5. What is an exonuclease? How do 5’ to 3’ and 3’ to 5’ exonucleases differ from one another functionally? How does an endonuclease differ from an exonuclease? An exonuclease is an enzyme that degrades nucleotides from the one end of the molecule which is different than an endonuclease which degrades nucleotide molecules from with in the molecule. The difference is on which strand the exonuclease is acting on. DNA polymerase III, for example, proofreads the template strand in the 3’5’ direction but lays new nucleotides in the 5’3’ direction. RNase acts as an exonuclease, deleting RNA primer in the 5’3’ direction on the leading/lagging strands. 6. Define the roles of each of the following in DNA replication: Helicase, DNA Polymerase I, DNA Polymerase III, Primase, Single Strand Binding Protein, Topoisomerase, DNA Ligase, RNAse. Helicase: unwinds parental double helix at the replication forks DNA Polymerase I: replaces the RNA nucleotides of the RNA primer with DNA nucleotides. DNA Polymerase III: adds new DNA nucleotides to the RNA primer that must first be laid down Single Strand Binding Proteins: Binds to and stabilizes singlestranded DNA until it is used as a template to prevent the just unwound helix from coming back together Topoisomerase: Relieves overwinding strain ahead of replication forks by breaking, swiveling, and rejoining DNA strands DNA Ligase: Joins okazaki fragments of lagging strand once the RNA primer is replaced with DNA nucleotides. On the leading strand, it joins the 3’ end of DNA that replaces primer to the rest o the leading strand of DNA RNase: an exonuclease that degrades the RNA primer before DNA polymerase I can replace the nucleotides 7. What’s the difference between the leading strand and the lagging strand? Why is it necessarily true that every replication bubble will have two leading strands and two lagging strands? The difference between the leading strand and the lagging strand is that the leading strand is continuously formed once the primer is laid down, DNA polymerase III moves towards the replication fork adding nucleotides. The lagging strand must wait as the double helix is unzipped because nucleotides can only be added on the 3’ end causing the DNA polymerase III to add nucleotides segment by segment in the opposite direction of the replication fork. There are two of each strand because replication occurs in a bidirectional format from the origin of replication creating two replication forks. 8. Although both catalyze new covalent bond synthesis in DNA, DNA Polymerase requires no ATP for energy, whereas DNA ligase does require the energy of ATP. Explain why this is so. This is because hydrolysis can drive the connection of new nucleotides to a growing strand by DNA polymerase but to connect a sugarphosphate backbone of a 3’ carbon to a 5’ carbon the the strand “in front” of it requires ATP to create the new bond. 9. Define the end replication problem in eukaryotes, and explain how eukaryotic cells deal with the problem. Replication machinery cannot complete the 5’ ends of daughter DNA strands because a DNA polymerase can add nucleotides only to a 3’ end of a preexisting polynucleotide. Once a primer is removed from the last fragment there is no 3’ end for new nucleotides to be added so repeated rounds of replication produce shorter and shorter DNA molecules with staggered ends. Eukaryotic chromosomal DNA molecules have special sequences called telomeres at the end that do not contain genes but multiple repetitions of one short nucleotide sequence. Telomeres prevent the cell from activating mechanisms to handle damaged DNA such as cell cycle arrest or apoptosis. Also the telomeric DNA acts as a buffer so the DNA does not shorten too fast. An enzyme called telomerase catalyzes the lengthening of telomeres in eukaryotic germ cells, thus restoring their original length and compensating for the shortening that occurs during DNA replication. 10. What is the Hayflick limit? How is it related to telomeres, cellular aging and senescence? The Hayflick limit is the number of times a normal human cell population will divide until cell division stops This is related to telomeres, cellular ageing, and senescence in that telomeric DNA tends to be shorter in dividing somatic cells of older individuals and in cultured cells that have divided many times. It has been proposed that shortening of telomeres is somehow connected to the ageing process of certain tissues and even to aging of the organism as a whole. Once a telomere is too short the cell will be signaled for apoptosis or senescence which is no more cell division leading to aging tissues/aging organism. 11. What is meant by the terms deamination and depurination? How does the cell “deal with” each of these problems? Deamination is the removal of an amine group from a molecule. Enzymes that catalyze this reaction are called deaminases. In DNA, deamination of cytosine is corrected by the removal of uracil. This site is then recognized by endonucleases that break a phosphodiester bond in the DNA, permitting the repair of the resulting lesion by replacement with another cytosine. A DNA Polymerase may perform this replacement by its 5'>3' exonuclease activity, followed by a fillin reaction by its polymerase activity. DNA ligase then forms a phosphodiester bond to seal the resulting nicked duplex product, which now includes a new, correct cytosine. This is done is similar fashion for all nucleotides Depurination occurs when a purine (A or G) is released from a sequence. The cell can repair this with an endonuclease that cuts a segment of DNA and then DNA polymerase rewrites the sequence to insert the missing base pair. 12. What is an intercalating agent? How do intercalating agents lead to mutation and DNA damage? Such as ethidium bromide and proflavine, are molecules that may insert between bases in DNA, causing frameshift mutation during replication. Frameshift mutation can lead to cancer cell growth or cause the cell to create proteins in incorrect order once transcribed to RNA and translated by ribosomes 13. How does exposure to ultraviolet radiation lead to DNA damage and potentially to mutations? UV is a physical mutagen known as mutagenic radiation which can cause disruptive thymine dimers in DNA. This new structure no longer hydrogenbonds to adenine, and creates a noticeable kink that will signal a base mismatch. If left to its own devices, DNA polymerase will “idle” at this site. The other side can be replicated, leaving a gap at the dimer. This would be lethal in the next round of replication, because it would produce a doublestranded break. 14. Define the four essential steps that excision repair pathways in both prokaryotic and eukaryotic cells have in common. Teams of enzymes detect and repair damaged DNA, such as a thyamine dimer (caused by UV radiation) which can distort a DNA molecule. A nuclease enzyme cuts the damaged DNA strand at two points, and the damaged section is removed. Repair, synthesis by a DNA polymerase fills in the missing nucleotides. DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete. 15. Why is cancer the logical consequence of damage to DNA repair mechanisms like the UV Repair pathway? Skin cancer can be caused by an inherited defect in a nucleotide excision repair enzyme because this can cause mutations to go unnoticed by the cell. The formation of thyamine dimers like those in UV damage caused DNA to buckle and interfere with DNA replication and if these go uncorrected, skin cancer can result. 16. How do some chemotherapeutic strategies exploit defective DNA repair mechanisms in cancer cells? Some chemotherapeutic strategies target the DNA repair and replication mechanisms in cancerous cells to prevent them from repairing DNA that has mutations and continuing to replicate it once repaired. Many cancerous cells do not have a replication limit and they will continue to divide and move mutated DNA to new cells spreading cancer. 17. Compare the enzyme responsible for transcription to the enzyme responsible for initiating each new nucleic acid strand in replication and the enzyme that does the bulk of DNA replication. How are the enzymatic activities similar? How are they distinct? RNA Polymerase II and DNA Polymerase III are similar in that they both read template DNA in the 5’3’ direction. The enzymes are different in that RNA Pol can initiate new nucleic acids whereas DNA Pol must have an RNA primer laid down first. RNA Pol also requires general transcription factors to initiate the transcription of mRNA. 18. What is the relationship between the sequence of the transcribed RNA molecule and the template and nontemplate strand of the gene? The mRNA molecule that is transcribed comes from an RNA polymerase II enzyme reading the template strand and producing a complimentary strand. The nontemplate strand is inside the RNA polymerase enzyme, however, it is not read to create an mRNA strand. 19. During transcription, where does the energy for the synthesis of the RNA molecule come from? How does this necessarily constrain the direction of synthesis of the transcribed RNA? How is this related to the synthesis of DNA during replication? This is the same as the construction of a new DNA molecule during replication in that nucleotides are added to the 3’ end of the RNA molecule via hydrolysis reactions. Only sugarphosphate backbone molecules can be attached at the 3’ carbon of the last molecule. 20. What is meant by coding versus noncoding RNA? What classes of RNA fall into each category and what are their major functions? Coding RNA refers to triplet nucleotide sequences that will code for a protein. Noncoding RNA consist of nucleotide sequences that will not be translated into proteins but have other important functions. mRNA: (coding) strand that is read by ribosome known as “messenger” RNA. Molecule of nucleotides that is transcribed from DNA and translated into AAs via ribosomes. tRNA (noncoding) Transfer RNA used to transfer amino acids from the cytoplasmic pool to a growing polypeptide in a ribosome. rRNA (noncoding) RNA molecules that, together with proteins, make up ribosomes; the most abundant type of RNA snRNA (noncoding) Small nuclear RNA that, together with proteins, make up spliceosomes miRNA (noncoding) Micro RNA that regulates the expression of genes by interacting directly with DNA or w mRNA copy. Capable of binding to complementary sequences in mRNA molecules. Allows complex to bind to any mRNA molecule with at least 7 or 8 nucleotides of complementary sequence. miRNA and protein complex either degrades the target mRNA or blocks its translation 21. What is meant by the term ribozyme? What classes of RNA are categorized as such and what are their major functions? Ribozymes are RNA molecules that function as enzymes. Intron RNA functions as a ribozyme and catalyzes its own excision. (rRNA) RNA is singlestranded, a region of an RNA molecule may basepair, in an antiparallel arrangement, with a complementary region elsewhere in the same molecule; this gives the molecule a particular 3D structure. Specific structure is essential to the catalytic function of ribozymes, just as it is for enzymatic proteins. Some of the bases in RNA contain functional groups that can participate in catalysis. Acid molecules add specificity to its catalytic activity 22. What distinguishes eukaryotic RNA Polymerase II from Pol I, III, IV and V? RNA polymerase I synthesizes a prerRNA which matures and will form the major RNA sections of the ribosome. RNA polymerase II synthesizes precursors premRNA. It controls transcription for the most part and a range of transcription factors are required for its binding to promoters. RNA polymerase III synthesizes tRNAs, rRNA and small RNAs found in the nucleus and cytosol. RNA polymerase IV synthesizes siRNA in plants. RNA polymerase V synthesizes RNAs involved in siRNAdirected heterochromatin formation in plants. 23. Why are general transcription factors required for transcription by Pol II? What are the major functions of the two general transcription factors we discussedTFIID(TBP) and TFIIH? Transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after tx factors are attached to the promoter does RNA polymerase II bind to it. TFIID/TBP: TATA binding protein that recognizes the promoter sequence and recruits general transcription factors to bind and assemble TFIIH: A helicase enzyme that unwinds parent DNA and phosphorylates RNA polymerase II at the Cterminal domain. Phosphates become binding sites for “hitchhiker” proteins such as Capping Enzymes and Cleavage factors. Also used as a promoter clearance 24. In general terms, how do the functions of general transcription factors like TFIID and regulatory transcription factors differ? General tx factors are essential for transcription of all proteincoding genes. A few general tx factors bind to a DNA sequence such as the TATA box with in the promoter but most bind to proteins, including other tx factors and RNA pol II. Regulatory tx factors determine the rate of gene expression. Tx factors bind to activation domains that bind other regulatory proteins and tx machinery, facilitating a series of proteinprotein interactions that result in enhanced transcription off a given gene. Some tx factors act as repressors and can inhibit gene expression in several ways (directly binding to control element of DNA, blocking activator binding or interfering with the activator itself so it can’t bind to the DNA) 25. What is a promoter? What is a TATA box and why is it called that? A promoter is the DNA sequence where RNA Pol II binds to begin transcription. The TATA box is a crucial sequence of DNA that is part of the promoter sequence where RNA polymerase binds. It is called this because the sequence is just that TATA. 26. What is the polyA signal sequence? What role does this sequence play in terminating transcription of a eukaryotic gene? What is its relationship to the polyA tail of eukaryotic mRNAs? The polyA signal sequence refers to the sequence TTATTT on DNA that is seen at the 5’ end of the template strand that is transcribed in the 3’5’ direction. When transcribed into mRNA it reads AAUAAA and is the sequence recognized by cleavage facts to end the transcription to create the premRNA strand. The polyA tail is added to the 3’ end of the newly transcribed mRNA by polyA polymerase to increase efficiency of translation and to slow degradation 27. What is the 5’ cap on eukaryotic mRNA? What is its functional significance? The 5’ cap on mRNA is a methylated Guanine nucleotide that is added immediately after tx begins. The cap protects the mRNA from exonucleases and is the initiation of translation for the ribosome to bind. 28. What are exons and introns? What molecular machine is responsible for recognizing these sequences and controlling their processing in eukaryotic mRNAs? Exons are regions of mRNA that are expressed by being translated into AAs and Introns are regions of mRNA that are noncoding segments that are usually between coding regions and are cut out by large complex of proteins and snRNA called spliceosomes. Transcription factors that bind to a DNA sequence, other transcription factors, and RNA polymerase II facilitate the correct positioning of the DNAenzymetx factor complex on the promoter sequence of DNA and initiate transcription (RNA synthesis). The interactions of enhancers also affect the rate of gene expression by binding to activators or repressor proteins that in turn interact with the transcription initiation complex. It is the combinatorial code of all these things that regulate transcription. 29. What advantage does alternative splicing confer to eukaryotic cells? This is one of several opportunities for regulating gene expression. Different mRNA molecules are produced form the same primary transcript depending on which RNA segments are treated as exons and which as introns. Regulatory proteins specific to a cell type control intronexon choice by binding to regulatory sequences within the primary transcript. This expands the repertoire of a eukaryotic genome. The extent of alternative splicing greatly multiplies the number of possible human proteins, which may be better correlated with the complexity of the organism. 30. What is the relationship between the exon and a “functional domain” on a protein? How is this related to the concept of protein evolution over time? Different exons code for the different domains of a protein including the functional domains of the active sites and cell membrane binding sites. The presence of introns in a gene may facilitate the evolution of new and potentially beneficial proteins as a result of a process known as exon shuffling. Introns increase the probability of crossing over between the exons of alleles of a gene simply by providing more terrain for crossover without interrupting coding sequences. This might result in new combos of exons and proteins with altered structure and function. The occasional mixing and matching of exons between different genes could occur and this may also lead to new proteins with novel combinations of functions. 31. What are the four fundamental properties of the genetic code? Genetic code is written in triplets: 3 nucleotides in mRNA known as codons specify 1 amino acid. Codons are redundant so that each amino acid (except Met and Trp) encoded by more than 1 codon. Codons are unambiguous; each codon specifies only 1 amino acid “Universal” coding for all organism that use essentially the same codes for amino acids 32. What are the two “business ends” of a tRNA molecule? What is their functional significance in translation? The two business ends include the Amino acid attachment site which uses the AARS to attach an amino acid to the tRNA and the Anticodon end which has the complimentary nucleotides to the mRNA being translated. 33. How is it that the process of linking tRNAs to their cognate amino acids is a relatively high fidelity process, given the similarity in structure between some amino acids, for example leucine and isoleucine? The active site of each type of AARS fits only a specific combination of amino acid and tRNA. There are 20 different synthetases, one for each amino acid. Each synthetase is able to bind to all the different tRNAs that code for its particular amino acid. The synthetase catalyzes the covalent attachment of the amino acid to its tRNA in a process driven by the hydrolysis of ATP. 34. Describe the structure of the eukaryotic (80S) ribosome, what role is played by the large and small subunits of this molecular complex? Small subunit Binds to both mRNA and a specific initiator tRNA, which carriers the AA methionine. Binds the mRNA at a specific RNA sequence, just upstream from the start codon, AUG. It binds to the 5’ cap of the mRNA and then moves, or scans, downstream along the mRNA until it reaches the start codon Large subunit Completes the initiation complex. Hydrolysis of GTP provides the energy for the assembly. Initiator tRNA is in the P site; the A site is available to the tRNA with the next AA 35. What do the “E”, “P” and “A” stand for in describing the three tRNA binding sites within the large ribosomal subunit? Describe the cycling of tRNAs through these three sites during the process of translation. What is happening to the mRNA molecule as this is occurring? The P site (peptidyltRNA binding site) holds the tRNA carrying the growing polypeptide chain while the A site (aminoacyltRNA binding site) holds the tRNA carrying the next amino acid to be added onto the chain. Discharged tRNAs leave the ribosome from the E site (exit site). The positions of the ribosome hold the tRNA and mRNA in a close proximity and positions the new amino acid so that it can be added to the carboxyl end of the growing polypeptide. It then catalyzes the formation of the peptide bond. 36. What is the general role of initiation factors in translation? What roles are played by elongation factors? What role is played by release factors? Initiation factors are required to bring all the components of translation together. The cell also expends energy obtained by hydrolysis of a GTP molecule to form the initiation complex. Elongation factors are required by the addition of amino acids to the growing polypeptide chain. The mRNA is moved through the ribosome in one direction (same as saying ribosome moves 5’3’ down RNA). Codon recognition requires hydrolysis of one molecule of GTP, which increases the accuracy and efficiency. One more GTP is hydrolyzed to provide energy for the translocation step. Release factors bind directly to the stop codon in the A site. The release factor causes the addition of a water molecule instead of an amino acid to the polypeptide chain. This reaction breaks the bond between the completed polypeptide and the tRNA in the P site releasing the chain through the exit tunnel of the ribosomes large subunit. 37. How does a given mRNA direct protein synthesis by either free ribosomes in the cytoplasm, or bound ribosomes at the rough ER? Polypeptide synthesis begins on a free ribosome in the cytosol. A signalrecognition particle binds to the signal peptide which targets the protein to the ER. The SRP binds to a receptor protein complex that forms a pore and has a signalcleaving enzyme. The SRP leaves and polypeptide synthesis resumes, with simultaneous translocation across the membrane. The signal cleaving enzyme cuts off the signal peptide. The rest of the completed polypeptide leaves the ribosome and folds into tits final conformation. 38. What is meant by the terms silent, missense, nonsense and frameshift mutation? How can a base change in a protein coding gene that causes a single amino acid substitution have either no effect, minimal effect or devastating effect on the protein function? Point mutations are changes in a single nucleotide causing the following mutations: o Silent mutation is a change in a nucleotide pair that may transform one codon into another that is translated into the same amino acid and there is no observable effect on the phenotype. o Missense mutations are substitutions that change one amino acid to another. This mutation may have little effect on the protein because the new amino acid has similar properties as the one it replaced or it may be in a region of the protein where the exact sequence of amino acids is not essential to the proteins function. This may be detrimental if the new amino acid is substantially different than the one it replaced or if the region in the protein complex is sensitive to changes. o Nonsense mutations occur when a point mutation to a nucleotide changes an amino acid to a stop codon and it causes translation to be terminated prematurely; the resulting polypeptide will be shorter than the polypeptide encoded by the normal gene. These usually lead to nonfunctional proteins. Insertion and deletions are additions or losses of nucleotide pairs in a gene. These have devastating effects on the resulting proteins and the organism as a whole. o Frameshift mutations are the alterations of the reading frame of the genetic message, the triplet grouping of nucleotides on the mRNA that is read during translation. All nucleotides downstream of the deletion or insertion will be improperly grouped into codons and will result in extensive missense usually ending sooner or later in nonsense and premature termination. 39. How could a mutation within an intron lead to disruption of protein function? What percentage of human diseases are thought to be the result of this type of mutation? A mutation in an intron may disrupt protein function because a mutation in an intron would cause a different site for RNA splicing. This leads to different outcomes of exons which ultimately serve as the coding regions of mRNA. 40. What is the molecular explanation for the diauxic growth curve of a culture of E. coli grown in the presence of glucose and an alternate sugar like lactose? The biphasic bacterial growth in the presence of glucose and alternate sugars like lactose is due to the preferred sugar by E. coli. A shift then occurs in the expression in genes to metabolize the next sugar. Bacteria utilize the glucose first because more energy from the monosaccharide than having to isomerize and split the disaccharide lactose. 41. What are the functions of the three structural genes of the lac operon of E. coli? Which, if any, of the three genes are indispensable for normal lactose metabolism? Lac Z: encodes the lactose betagalactosidase which cleaves and begins metabolism of lactose. Lac Y: encodes for lactose permease which is a membrane transport protein Lac A: (dispensible) encodes galactosidase transacetylase enzyme which is not understood completely 42. Distinguish between the promoter, operator and CAP binding sites of the lac operon. Where are they located relative to the structural genes and to one another? Which of these are considered “regulatory elements”? What are they regulated by? Why is the I gene expressed independently of the lac operon structural genes? Promoter directs RNA polymerase to bind to gene and begin transcription located upstream next to operator and upstream from the +1 location. Operator is the binding site for the regulatory protein that negatively regulates transcription known as a repressor. It is located next to +1 and the lac Z gene. CAP Site is where the regulatory protein CAP binds which is located next to and upstream from the promoter. CAP protein interacts with RNA polymerase II to stabilize promoter because the promoter is “weak” without it. The CAP site is considered regulatory because when cAMP binds to the CAP protein it is because glucose is not present. This occurs because glucose will block adenylyl cyclase from catalyzing cAMP. The I gene is expressed independently because it encodes for a repressor regulatory protein. This repressor will bind to the operator site when no lactose is present because there is no allolactose to allosterically inhibit the repressor from binding. 43. Why is the lactose operon referred to as an inducible operon? What is the molecular mechanism of induction? How does this mechanism effectively allow the E. coli genome to “sense” when lactose is present? The lac operon is referred to as inducible because it is usually off but can be stimulated when a specific small molecule interacts with a regulatory protein. The regulatory protein is the, usually inactive, repressor protein that gets activated in the presence of allolactose (lactose). 44. What is the molecular mechanism that leads to higher levels of lac operon transcription when glucose is absent? How does this mechanism effectively allow the E. coli genome to “sense” when glucose is present? When glucose is not present cAMP concentrations can increase because its catalysis is not inhibited by the presence of glucose. The cAMP allosterically activates CAP which can bind to the CAP site on the operon. When CAP binds it increases the transcription rate of RNA polymerase II. It acts as a sensor for the presence of glucose because if glucose where present, concentrations of cAMP would be lower and CAP would be less likely to be activated. 45. Generally speaking, what “types” of eukaryotic genes are regulated and what types would you expect to be expressed constitutively over the life of the cell? Why? Constitutive genes are not regulated and may code for proteins that are necessary for normal cell function such as cell membrane proteins, organelles, etc. Facultative genes are regulated as needed such as genes signaling rapid cell replication as seen during embryonic development. 46. What functional distinction can be made between general transcription factors like TFIID and gene specific transcriptional activators and repressors? General transcription factors are used by both eukaryotes and prokaryotes; regulatory transcription factors are used by eukaryotes. General tx factors like TFIID are required for transcription
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