BIOL 3000, Exam 3 Study Guide
BIOL 3000, Exam 3 Study Guide BIOL 3000
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This 23 page Study Guide was uploaded by Jerry Garcia on Monday May 9, 2016. The Study Guide belongs to BIOL 3000 at Auburn University taught by Dr. Graze in Spring 2016. Since its upload, it has received 4 views. For similar materials see Genetics in Biology at Auburn University.
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BIOL 3000 Genetics Exam III Study Guide Information in italics is there for clarification, but I do not expect you to memorize terms or concepts that are in italics. Proteins and motifs you need to know are in bold. If in doubt, ask. Chapter 9: DNA Replication Proteins Involved in Replication: 1. Initiator proteins: recognizes/binds the origin of replication, unwinds a short stretch of DNA, allows helicase and other singlestrandedbinding proteins to attach to the polynucleotide strand, and allows replication to be initiated (for example, DnaA in prokaryotes) 2. DnaA (prokaryotic): binds to the DnaA box (DnaA motif) sequences and forms a balllike complex that bends DNA, causing the strands in the ATrich regions to separate into an open bubble 3. Singlestrandedbinding proteins (SSBs): tetramer proteins that bind and stabilize the ssDNA, keeping it from base pairing with itself and or the other strand 4. DNA gyrase: member of the family of topoisomerases; reduce supercoiling of DNA and release torque buildup ahead of replication fork 5. Helicase (DnaB in prokaryotes): breaks hydrogen bonds in order to open dsDNA strands, making them ssDNA 6. Primase: lays down an RNA primer, providing a 3’OH group for the attachment of a DNA nucleotide at the initiation of replication 7. DNA polymerase(s): bind RNA primer at 3’OH and makes new strands/synthesizes DNA; 5’ to 3’ polymerase activity; 5’ to 3’ exonuclease activity to remove the RNA primer; 3’ to 5’ exonuclease activityerror correction a. DNA polymerase III (prokaryotes): 5’ to 3’ polymerase activity for synthesis, 3’ to 5’ exonuclease activity (think erasing) to correct errors b. DNA polymerase I (prokaryotes): 5’ to 3’ polymerase activity for synthesis, 3’ to 5’ exonuclease activity (think erasing) to correct errors, 5’ to 3’ activity to remove primers (then fills them in) c. DNA polymerase alpha (eukaryotes): Initiation of synthesis through priming activity in conjunction with primase and lays down ~2030 bases of DNA after the RNA primer; 5’ to 3’ polymerase activity; low processivity d. DNA polymerase delta (eukaryotes): 5’ to 3’ polymerase activity for lagging strand synthesis, primer displacement, 3’ to 5’ exonuclease activity; high processivity e. DNA polymerase epsilon (eukaryotes): 5’ to 3’ polymerase activity for leading strand synthesis, 3’ to 5’ exonuclease activity; high processivity 8. Ligase: joins together the newly synthesized strands of DNA by sealing, catalyzing phosphodiester bonds 9. Telomerase: ribonucleoprotein containing an RNA that is complimentary to Grich repeats (G overhang) that make up telomeres. It binds to those repeats, creating DNA sequence complimentary to the RNA template in the protein and extending the 5’ end of the chromosome in the process (building telomeres). Steps in Replication: Initiation: recognizes and opens DNA at origins of replication Prokaryotic DnaA recognizes DnaA motif (DnaA box) at OriC and many copies of this initiator protein bind. A coil forms around these proteins and causes the DNA to open in the ATrich region. The helicase (DnaB and its cofactor) can then bind to the open bubble/complex and start to unwind the DNA. Eukaryotic multiple origins that consist of AT rich sequence but differ from prokaryotic origins. The prereplication complex (preRC) [preRC=ORC + replication licensing factors) forms at the origin starting with formation of the origin recognition complex (ORC) (binds to origin first) and binding of replication licensing factors (binds after ORC). Additional proteins bind and load the helicase (MCM complex). Replication is only initiated at licensed sites. Origins fire only once per cell cycle because the licensing factors are active and present only prior to initiation and are not active after replication is initiated. Unwinding: pull apart the two strands in dsDNA helix Requires helicase activity, in prokaryotes the helicase is a hexameric protein consisting of 6 DnaB units; in eukaryotes the MCM complex which is part of the prereplication complex is responsible for helicase activity. The dsDNA is unwound producing a replication bubble made of open ssDNA. Unwinding also requires topoisomerase activity unwinding makes the DNA super coiled (tightly twisted). Gyrase undoes the twists by breaking and resealing DNA to remove twists. Without this you would get uncontrolled ‘knotting’ as the two strands are pulled apart. Elongation: continued synthesis of new DNA as replication bubble continues to open DNA polymerase synthesizes the new strand, but requires a 3’ OH to initiate synthesis. First Primase recognizes origin points, binds to ssDNA, and lays down an RNA primer. Primase does not require 3’ OH to start synthesis. It synthesizes 10 – 12 bases (prokaryotes) of RNA, to provide the 3’ OH. In eukaryotes, the primer consists of RNA plus ~2030 bp DNA. DNA polymerase then synthesizes new strands of DNA from an ssDNA template from 5’ 3’ – this is a huge complex (a holoenzyme). After synthesis on the lagging strands ligase joins the nicks in the sugarphosphate backbone between Okazaki fragments. Termination: In E. coli, a protein (Tus) binds to termination sequences and stalls the replication fork and the holoenzyme ‘falls off’ the DNA. The remaining nick in the circle is ligated. In eukaryotes, the end of the chromosome is reached. The primer is removed, but DNA polymerases can’t fill the overhang with new DNA sequence because there is no 3’OH. If this was the last step when those strands were replicated the 5’ ends would be reduced by approximately the length of a primer. Instead telomerase binds to Grich repeats at chromosome ends. It provides an RNA template for step by step extension of the strand with the 3’ end. This makes a long overhang consisting of repeats of sequence complimentary to the RNA in the telomerase (a ribonucleoprotein and reverse transcriptase DNA from an RNA template). The extended overhand gets filled in, it is thought that an RNA primer is laid down and then the gap is filled again leaving a short overhang (but not shortening the overall length of the chromosome). Definitions: 1. Primer: short stretch of RNA on a DNA template; provides a 3’OH group for the attachment of a DNA nucleotide at the initiation of replication 2. Proofreading: ability of DNA polymerase to remove and replace incorrectly paired nucleotides in the course of replication 3. Motif: functional DNA sequence 4. Consensus sequence: most commonly observed base (A/T/G/C) at each position in a functional sequence *Other definitions/concepts included above* Study Guide Questions: 1. Given a diagram of a replication fork, including proteins bound to the DNA in different locations, identify different proteins (labeled in the diagram with A, B, C, D) and their corresponding functions. 5 ’ 3 1) Singlestrandedbinding proteins: stabilize ssDNA 2) Gyrase: reduce supercoiling ahead of replication fork 3) Helicase: breaks hydrogen bonds and opens dsDNA at replication fork 4) Primase: lays down a primer 5) Ligase: seals Okazaki fragments between removed primers 6) DNA polymerase: synthesizes DNA in the 5’ to 3’ direction 2. Label a diagram showing how telomeres at the ends of eukaryotic chromosomes are replicated, indicating the telomerase, the template, DNA, RNA, 5’ and 3’ ends. Chapter 10 Part I: Transcription Basic Rules of Transcription: Transcription is a selective process Only certain parts of DNA are transcribed RNA is transcribed from ssDNA Within a gene only one strand is normally transcribed RNA messages are antiparallel and complementary to the DNA template strand Transcription is always in the 5’3’ direction Transcription depends on RNA polymerase (RNA Pol) RNA Pol is a complex, multimeric (many proteins) enzyme complex Promoters contain short sequences (motifs) critical in the binding of RNA Pol to the DNA strand RNA Pol does not require a primer to start synthesis Complex elements of transcription: Promoters vary, for example some promoters have only the TATA box or only the DPE and some have both. For example, in humans 25% of genes have a TATA box and only 10% have the canonical sequence (TATAAA) exactly. Promoters can have varying ‘strength’ related to how well different factors bind (and to differences in their motifs from the canonical sequences). The basal transcription rate of different genes can therefore be different. Many promoters start transcription via initiation and then the RNAPII holoenzyme pauses after a short piece is transcribed. It remains paused until additional activators release the pause. Definitions: 1. Ribozyme: RNA molecule that can act as a biological catalyst 2. Ribosomal RNA (rRNA): RNA molecule that is a structural component of the ribosome; ribosome function (part of the ribonucleoprotein complex) 3. Messenger RNA (mRNA): RNA molecule that carries genetic information for the amino acid sequence of a protein 4. Premessenger RNA (premRNA)/Heterogeneous RNA (hnRNA): eukaryotic RNA molecule that is modified after transcription to become mRNA 5. Transfer RNA (tRNA): RNA molecule that carries an amino acid to the ribosome and transfers it to a growing polypeptide chain in translation (i.e. transports amino acids) 6. Small nuclear ribonucleoprotein (snRNP): structure found in the nuclei of eukaryotic cells that consists of small nuclear RNA (snRNA) and proteins; functions in the processing of premRNA 7. Template strand: the strand of DNA that is used as a template during transcription; the RNA synthesized during transcription is complementary and antiparallel to the template strand 8. Nontemplate strand: the DNA strand that is complementary to the template strand; not ordinarily used as a template during transcription 9. Promoter: DNA sequence to which the transcription apparatus binds so as to initiate transcription; indicates the direction of transcription, which of the two DNA strands is to be read as the template, and the starting point of transcription; two types: core and proximal 10. Terminator: sequence of DNA nucleotides that causes the termination of transcription 11. Ribonucleoside triphosphates (rNTPs): substrate of RNA synthesis; consists of ribose, a nitrogenous base, and three phosphates linked to the 5’carbon atom of the ribose; in transcription, two of the phosphates are cleaved, producing an RNA nucleotide 12. RNA polymerase: enzymes that synthesize RNA from a DNA template during transcription 13. Core enzyme: part of the bacterial RNA polymerase that, during transcription, catalyzes the elongation of the RNA molecule by the addition of RNA nucleotides; consists of four subunits: two copies of alpha, a single copy of beta, and a single copy of beta prime 14. Sigma factor: subunit of bacterial RNA polymerase that allows the RNA polymerase to recognize a promoter and initiate transcription 15. Holoenzyme: complex of an enzyme and other protein factors necessary for complete function (sigma factor plus the core enzyme form a holoenzyme) 16. RNA polymerase II: eukaryotic RNA polymerase that transcribes premRNA, some snRNAs, and some miRNAs 17. Exon: contain the code needed to make proteins 18. Intron: “intervening sequences”, noncoding areas of genes that do not generally impact genotype 19. Codon: sequence of three nucleotides that encodes one amino acid in a protein 20. 5’ Untranslated region (5’ UTR): aka the leader, a sequence of nucleotides at the 5’ end of the mRNA that does not encode any of the amino acids of a protein 21. 3’ Untranslated region (3’ UTR): aka the trailer, a sequence of nucleotides that is at the 3’ end of the mRNA and is not translated into protein 22. ShineDalgarno sequence: consensus sequence found in the bacterial 5’ UTR of mRNA and contains the ribosomebinding site 23. Proteincoding region: the part of mRNA consisting of the nucleotides that specify the amino acid sequence of a protein Proteins involved in transcription (eukaryotic transcription will be the focus): 1. General Transcription Factors: required for transcription and bind to core promoter; all genes 2. TBP (TATA Binding Protein): essential component of TFIID that recognizes and binds to the TATA box 3. TFIID: Binds at the promoter and recruits other general transcription factors that are required for recognition of the core promoter by RNA pol II 4. TFIIB: binds to the BRE element. Involved in initiation, stabilizes the TFIID complex and is involved in recruiting RNA pol II (complexed with other general transcription factors) to the core promoter 5. Mediator complex: part of the holoenzyme that interacts with transcription factors 6. RNA Pol II core enzyme: is a multisubunit enzyme that catalyzes the synthesis of RNA from a ssDNA template. The core enzyme contains subunits necessary for polymerization of RNA molecules 7. RNA Pol II holoenzyme: Transcription requires RNA Pol II AND the general transcription factors (for example TFIIB, TFIIE, TFIIF) + more. The holoenzyme contains all subunits necessary to begin transcription, includes the CTD tail, general transcription factors and mediator complex. Note, the CTD tail is part of the holoenzyme and phosphorylation of the tail is a signal for initiation and elongation. The tail also plays a role in premRNA processing 8. TFIIH: Acts as a helicase during formation of the open complex. Also active in phosphorylating the CTD tail of RNA Pol II during initiation (allowing it to be released so that elongation can start). 9. Specific Activators/Transcription Factors: stabilize the holoenzyme, sometimes chromatin modification, specific for different genes (regulatory). 10. Elongation factors: required for high processivity (the ability to catalyze consecutive reactions, determines the length of nucleic acid that can be stably synthesized). Evict histones ahead of RNAP II and replace them behind it. Maintain heteroduplex RNA: DNA hybrid region. Deal with lesions and problematic sequence (resulting in bends or if there is complementary base pairing in the ssDNA etc.) that could stall or stop transcription 11. Termination factors: Involved in cleaving the RNA and release of RNAPII from the template. Steps of Transcription: 1. Open chromatin conformation such that the core promoter can be accessed by general TFs 2. Recognition: of the core promoter, binding of TBP to the TATA box (or nonspecifically if the gene does not contain a TATA box), TFIID complex is recruited (binds to TBP), followed by TFIIA and TFIIB (recognizes the BRETFIIB recognition element) and others. These proteins act in a complex to recruit/ allow RNA Pol II to bind at the core promoter. 3. Initiation: Formation of the preinitiation complexbinding of RNAPII complexed with general TFs, mediator complex + more = RNA Pol II holoenzyme. Interactions between holoenzyme and activators (specific TFs) in regulatory regions trigger initiation (change in protein conformation) which involves phosphorylation of the CTD tail by TFIIH (and sometimes the mediator complex) and release from the promoter. 4. Elongation: Elongation factors (pause and release for some promoters) + holoenzyme = RNA Pol II open complex (or elongation complex) moves along the DNA transcribing the RNA molecule. 5. Termination: STOP, RNAPII releases DNA, signal is cleavage of transcript and dephosphorylation of the RNAPII CTD tail also occurs. Study Guide Questions: Know the molecular definition of a gene. All the DNA sequences necessary to synthesize a functional RNA molecule and/or protein (we will focus on protein coding genes) Know the basic steps of transcription, be able to describe what happens at each step. *Answered above* Be able to match different types of RNA (for example tRNA) to definitions or steps of molecular processes. rRNA: structural and functional components of the ribosome; cytoplasm; bacterial and eukaryotic mRNA: carries genetic code for proteins; nucleus and cytoplasm; bacterial and eukaryotic tRNA: helps incorporate amino acids into polypeptide chain; cytoplasm; bacterial and eukaryotic snRNA: processing of premRNA; nucleus; eukaryotic snoRNA: processing and assembly of rRNA; nucleus; eukaryotic miRNA: inhibits the translation of mRNA; cytoplasm, eukaryotic siRNA: triggers the degradation of other RNA molecules; cytoplasm; eukaryotic Know the difference between general and specific transcription factors. General transcription factors bind to the CORE promoter near the start site and upstream of a gene and are part of the basal transcription apparatus that initiates transcription. Specific transcription factors (transcriptional regulator proteins) bind to the proximal (regulatory) promoter that stimulates and stabilizes the core promoter or upstream/downstream (distal) regulatory regions such as enhancers (bind activators) and silencers (bind repressors). Transcriptional regulator proteins/specific transcription factors are either activators (transcriptional activator proteins) that stimulate transcription, or repressors that inhibit transcription. Gene structure: a) Given a blank diagram of a typical eukaryotic gene structure, label the parts, including the following: Distal or proximal regulatory regions (5), not transcribed, noncoding The core promoter and one of the two ‘signature’ motifs highlighted (the TATA box or the BRE), by relative position to the +1 site and motif for the TATA box. Core promoter (1) not transcribed, noncoding; TATA Box (4) not transcribed, noncoding; BRE motif would be few nucleotides upstream of TATA Box The +1 site transcribed 5’ UTR (7), transcribed, noncoding Exons and Introns exons (3) transcribed and coding; introns (2) transcribed and noncoding 3’ UTR (8) transcribed, noncoding Transcription start site (6)? Transcribed, noncoding (within 5’ UTR) AAUAAA consensus sequence after 3’ UTR, transcribed b) Know the biological function of each of the parts. For example, be able to identify which parts of the gene contain protein coding information. *above* c) Identify which parts are transcribed and/or which are not. *above* Given a typical eukaryotic gene structure, identify where [TBP or TFIID or TFIIB or RNA polymerase II] bind, you will be given names of corresponding regulatory motifs, their positions and the +1 site. TBP and TFIID bind at the TATA box in the core promoter, TFIIB binds to the BRE motif in the core promoter, and RNA polymerase II binds at the INR and DPE at the core promoter downstream of the +1 site. Identify from several diagrams or descriptions the correct diagram or description that describes how specific transcription factors which bind enhancers far away from the core promoter can promote transcription. This may include labeling protein complexes in a blank diagram (e.g. identifying the mediator complex). Chapter 10 Part II: RNA Processing Definitions: 6. 5’ cap: modified 5’ end of eukaryotic mRNA, consisting of an extra nucleotide (methylated) and methylation of the 2’ position of the ribose sugar in one or more subsequent nucleotides; plays a role in binding of the ribosome to mRNA in translation and affects mRNA stability and the removal of introns 7. Poly (A) tail: string of adenine nucleotides added to the 3’ end of a eukaryotic mRNA after transcription 8. RNA splicing: process by which introns are removed and exons are joined together 9. 5’ splice site: the 5’ end of an intron where cleavage takes place in RNA splicing 10. 3’ splice site: the 3’ end of an intron where cleavage takes place in RNA splicing 11. Branch point: adenine nucleotide in nuclear preRNA introns that lies from 18 to 40 nucleotides upstream of the 3’ splice site 12. Spliceosome: large complex consisting of several RNAs and many proteins that splices protein encoding premRNA; contains five small ribonucleoprotein particles (U1, U2, U4, U5, U6) 13. Lariat: looplike structure created in the splicing of nuclear premRNA in which the 5’ end of an intron is attached to a branch point in premRNA 14. Alternative splicing: process by which a single premRNA can be spliced in more than one way to produce different types of mRNA 15. Cloverleaf structure: secondary structure common to all tRNAs 16. Anticodon: sequence of three nucleotides in tRNA that pairs with the corresponding codon in mRNA in translation 17. Large ribosomal subunit: the larger of the two subunits of a functional ribosome 18. Small ribosomal subunit: the smaller of the two subunits of a functional ribosome Proteins involved in RNA processing: 1. Capping enzyme: the guanylyl transferase that adds guanosine to the 5’ end of the pre-mRNA transcript 2. RNA methyltransferase: methylates the guanosine at position 7 and several downstream nucleotides 3. PAP (polyadenylate polymerase): the enzyme that adds the adenine nucleotides. 4. PAB (PolyA binding protein): promotes export from nucleus, translation and stability. 5. Cleavage Factors CPSF: Cleavage & polyadenylation specific factor (recognizes AAUAAA) CSF: Cleavage stimulation factor (recognizes G/U Rich Region) 6. The spliceosome: A large multicomponent complex (with changing configuration and membership) that catalyzes the reactions necessary for intron removal 7. snRNPs small nuclear ribonucleoproteins; have an snRNA component (U #) that functions in recognition and a protein component that functions in splicing; components of the spliceosome Steps of processing of protein coding eukaryotic genes: Addition of a 5’ 7methylguanosine cap The RNA molecule has a 5’ triphosphate. Capping enzyme is recruited to the RNAPII CTD tail and adds an inverted guanosine. This occurs after a short piece of RNA has been transcribed. RNA methyltransferase methylates the added nucleotide and one or more bases prior to that. The 5’ cap functions in initiation of translation Addition of a poly(A) tail (3’ end) The 3’ end of the mRNA transcript is cleaved (coupled to termination of transcription). Requires the AAUAA motif (after 3’ UTR) at the end of the transcript and a downstream G/U rich sequence. The cleavage factors cleavage & polyadenylation specific factor + Cleavage stimulation factor recognize the AAUAAA motif and the G/U Rich Region respectively. These bind and bend the transcript at the cleavage site. Additional cleavage factors CFI and CFII are recruited and a complex forms that cleaves the 3’ end of the transcript. Polyadenylate polymerase (PAP) must be bound for cleavage to occur, but does not itself catalyze the cleavage reaction. PAP adds a series of adenines to the 3’ end (~250). The poly(A) tail (with PolyA Binding proteins bound) functions in translation, in transport and in RNA stability (the lifespan of the RNA molecule after transcription .. transcripts degrade over time). Splicing removal of intronic sequences. Requires sequence motifs that bind snRNPs at the 5’ splice site, the branch point, a polypyrimidine tract and the 3’ splice site. o Two reactions: The 5′ splice site is attacked by the 2′OH of a conserved unpaired (not in a loop) adenosine (branch point), resulting in the formation of a 2′,5′phosphodiester linkage The free 3′OH group of the 5′ exon attacks the 3′ splice site, liberating the circular intron lariat and ligating exons together Sequence of events (concentrate on the sequence of events rather than the different snRNPs (e.g. U2), you need to know the names of the motifs and their role and that this process is mediated by snRNPs that are part of the spliceosome that have different U RNA elements which mediate interactions via complimentary base pairing) o U2af (an snRNP that is part of the spliceosome) binds to the pyrimidine tract and recruits U2. o U2 binds to the branch point via complimentary base pairing between the U RNA element and the sequence in the mRNA surrounding the branch point. o U1 binds to the 5’ splice site via base pairing between sequences in its U RNA and the sequence motif at the 5’ splice site. o U1 recruits the complex of U4, U5 and U6 to the 5’ splice site o A conformation change occurs (mediated by base pairing between snRNAs in the snRNPs) and U4 is kicked out (U1 might remain but is not active). o U2 and U6 form a complex bending the RNA molecule. The first reaction that is catalyzed results in cleavage at the 5’ splice site and attachment of the 5’ guanine to the adenine at the branch point (a 2’, 5’ phosphodiester bond), forming the lariat structure o The intron is cleaved from the exons in the second reaction. o U5 ligates the exons together Complex elements of RNA processing: There can be alternate 3’ cleavage sites and the poly(A) tail length can be modified at later stages Alternative splicing (different combinations of exons make alternative mRNAs from the same genetic information) happens and is regulated by proteins which bind in exons or introns and promote or inhibit splicing at a nearby site. Study Guide Questions: Know the three major differences between an mRNA and a premRNA and the functions of these modifications. PremRNA (hnRNA) is modified after transcription in the nucleus by the addition of a 5’ 7methylguanosine cap at the 5’ end of the transcript, the addition of a poly (A) tail at the 3’ end of the transcript of cleavage of the extra nucleotides at the end containing the G/U rich sequence, and splicing by spliceosomes that “cut out” the introns and leave only exons. The remaining mature mRNA contains the 5’ cap, the 5’ UTR (not translated into amino acids) at the beginning of the first exon, the proteincoding region with exons that gets translated into amino acids, the 3’UTR (not translated into amino acids) at the end of the last exon containing the AAUAAA consensus sequence, and the poly (A) tail at the 3’ end of the mature mRNA after cleavage of extra nucleotides past the 3’ cleavage site. Be able to identify the two unusual phosphodiester bonds that form during mRNA processing and match them to the corresponding step of RNA processing. 1) The 5’ to 5’ triphosphate bond during the addition of the 5’ 7methylguanosine gap 2) The 2’, 5’ phosphodiester bond between the 5’ guanine and the 2’OH of the adenine at the branch point during the formation of the lariat mRNA Processing o a) For a eukaryotic protein coding gene, match enzymes and/or descriptions of processing steps to the correct modification of the premRNA. 5’ Cap: Capping enzyme and methyl transferase Poly (A) tail: Cleavage factors, PAPs (polyadenylate polymerases), and PABs (poly A binding proteins) Intron/exon splicing: spliceosomes and snRNPs o b) Label the parts of an mRNA (e.g. 5’ UTR, start codon etc.), given the premRNA. o c) Steps of RNA processing are coordinated with transcription. Be able to identify which part of RNA pol II, if any, is required for this coordination. The CTD tail of RNA polymerase II is required for this coordination because it is where capping enzyme is recruited and adds an inverted guanosine after a short piece of RNA is transcribed. o d) Two of these processing steps produce modifications that are important for eukaryotic translation. Identify them and be able to provide a brief explanation of their role in translation. Addition of 5’ cap: Addition of poly (A) tail: Splicing o a) Given the structure of a typical eukaryotic premRNA molecule transcribed from a gene containing one intron and two exons, be able to identify the splice sites, branch point, and pyrimidine tract. o b) Identify the first [or second] reaction that is catalyzed by the spliceosome o c) What happens to the lariat after the two exons are ligated together? It is degraded o d) Splicing would be stopped at which of the following steps [initial recognition, reaction 1, reaction 2] if a mutation abolished the ability of components of the spliceosome to bind to the [5’ splice site, 3’ splice site, branch point or pyrimidine tract]? Explain would happen if [one of the RNA processing steps] failed to occur? Chapter 11: Translation Definitions: 1. Amino acid: repeating unit of proteins; consists of an amino group, a carboxyl group, a hydrogen atom, and a variable R group that is different for each amino acid 2. Peptide bond: chemical bond that connects amino acids in a protein 3. Polypeptide: chain of amino acids linked by peptide bonds 4. Degenerate genetic code: refers to the fact that the genetic code contains more information than is needed to specify all 20 common amino acids 5. Synonymous codons: different codons that specify the same amino acid 6. Wobble: base pairing between codon and anticodon in which there is nonstandard pairing, usually at the third (3’) position of the codon; allows more than one codon to pair with the same anticodon and allows different codons to specify the same amino acid 7. Nonoverlapping genetic code: refers to the fact that, generally, each nucleotide is part of only one codon and encodes only one amino acid in a protein 8. Initiation codon (start codon): the codon in mRNA that specifies the first amino acid (fMet in bacterial cells; Met in eukaryotic cells) of a protein; most commonly AUG 9. Stop codon (termination or nonsense codon): codon in mRNA that signals the end of transcription and does not code for an amino acid; the three common stop codons are UUA, UAG, and UGA 10. Universal genetic code: refers to the fact particular codons specify the same amino acids in almost all organisms 11. Aminoacyl tRNA synthetase: enzyme that attaches an amino acid to a tRNA; each aminoacyl tRNA synthetase is specific for a particular amino acid 12. tRNA charging: chemical reaction in which an aminoacyltRNA synthetase attaches an amino acid to its corresponding tRNA 13. Initiation factors: proteins required for the initiation of translation in bacterial cells 14. 30S initiation complex: initial complex formed in the initiation of translation in bacterial cells; consists of the small subunit of the ribosome, mRNA, initiator tRNA charged with fMet, GTP, and initiator factors 1, 2, and 3 15. 70S active ribosome (70S initiation complex): final complex formed in the initiation of translation in bacterial cells; consists of the small and large subunits of the ribosome, mRNA, and initiator tRNA charged with fMet 16. Aminoacyl/acetyl “acceptor” (A) site: one of the three sites in a ribosome occupied by a tRNA in translation; all charged tRNAs (with the exception of the initiator tRNA) first enter the A site in translation 17. Peptidyl “polypeptide” (P) site: in the elongation state of protein synthesis, tRNAs move from the aminoacyl (A) site into the P site 18. Exit (E) site: in the elongation stage of translation, the tRNA moves from the P site to the E site from which it then exits the ribosome 19. Elongation factors: proteins involved in the elongation of translation 20. Translocation: movement of a ribosome along mRNA in the course of translation 21. Release factors: protein required for the termination of translation; binds to a ribosome when a stop codon is reached and stimulates the release of the polypeptide chain, the tRNA, and the mRNA from the ribosome Features of the genetic code: 1. Written in a linear form (stored in the mRNA) 2. Coded as triplets (codons) 3. Unambiguous (1 codon = 1 amino acid ) 4. Degenerate (1 amino acid, AA, can be coded for by more than 1 codon) more info than needed 61 codons = 20 AAs) 5. Specific start and stop codons (Start – AUG Stop UAA, UGA, UAG ) 6. Commaless (there are no pauses in the code) 7. Nonoverlapping – ACCGCUGGG = only ACCGCUGGG and never read ACC, CCG, CGC etc. 8. Universal with rare exceptions (e.g., different termination codons) 9. There is wobble: a. 30 to 50 tRNAs and 61 codons = more codons than anticodons b. Different codons sometimes pair with the same anticodon c. Wobble = flexibility in base pairing at the third position of the codon d. If you look at the tables, many different codons differ only at the third position, they are called synonymous. For example, Alanine = GCU, GCC, GCA and GCG. Complexes, proteins, and RNAs to know: tRNA (transfer RNA): delivers amino acids one by one to the growing peptide chain at the ribosome. Has an “anticodon” at the tip that recognizes a 3nucleotide codon on the mRNA through complementary base pairing and an acceptor site (or stem) that becomes covalently bound to an amino acid. rRNA (ribosomal RNA): RNAs that are structural and enzymatic (ribozyme) components of the small and large subunits of the ribosome. Aminoacyl synthetase direct the charging of each tRNA by matching correct tRNAs to specific amino acids and catalyzing formation of a covalent bond between the adenine in the tRNA acceptor and an amino acid (20 different synthetases). Small Ribosomal Subunitthe part of the ribosome that recognizes mRNA transcripts and initiates translation. Large Ribosomal Subunitstabilizes the tRNAs and amino acids that are bound to anticodons and contains the ribozyme that catalyzes peptide bonds between amino acids. Initiation factors – IF3 prevents the large subunit from binding during initiation, IF2 brings the first tRNA to the Psite and a molecule of GTP. IF1 binds to the A site and blocks it. 30S initiation complex forms at the ShineDalgarno sequence positioning the ribosome over the start codon. Also required so that the first tRNA can be brought in at the P site and can pair with the start codon before the large subunit binds. 70S initiation complex A functional ribosome with an A (aminoacyl) site, P (peptidyl) site and E (exit) site, ready for elongation to begin. Elongation factorsEFTu brings a charged tRNA and a molecule of GTP to the A site of the ribosome. EFG is responsible for moving (translocation) the ribosome to the next codon (3 bases at a time). Peptidyl transferase activity (ribozyme) rRNA component of the large subunit that catalyzes formation of a peptide bond between an AA attached to the tRNA in the P site and the AA that is attached to the tRNA in the A site. Release factorsRF1 and RF2 recognize stop codons, binding at the A site instead of a tRNA, RF3 promotes cleavage of the polypeptide from the tRNA in the P site and destabilizes the ribosome triggering release of the tRNA from the P site, release of the mRNA and disassociation of the ribosome. Steps in translation (you need to know differences between eukaryotes and prokaryotes in initiation): Note: You do not need to memorize which steps need GTP, although it is included here for clarity as some factors bind GTP. Questions will provide the names of the individual initiation, elongation or release factors and some context as to the step being asked about. You need to know what each one does, but I will construct questions so that confusing, for example IF1 with IF3, will not be a problem. 1. tRNA Charging A charged tRNA has an AA linked to the tRNA, for transport to the ribosome. tRNAs are ssRNA that form a cloverleaf structure with an anticodon in the loop and a AA acceptor site at the 3’ end (CCA sequence). Aminoacyl synthetases catalyze the addition of an AA (COO group) to a tRNA at the 3’ end acceptor site. There is a different Aminoacyl synthetase for each of the 20 different AAs. This requires energy provided by cleaving ATP in the reaction: AA + tRNA + ATP > aminoacyltRNA + AMP + PPi. 2. Initiation components assembled The small ribosomal subunit becomes bound to the mRNA at the initiation site (start codon – AUG Methionine in eukaryotes or Nformylmethionine in prokaryotes). The charged tRNA with the specific anticodon for the start site, binds at the triplet in the P site of the small subunit. Following the association of the tRNA with the small subunit, the large subunit combines with the small to form the complete ribosome. In prokaryotes Initiation factors 3, 2, and 1 are involved in initiation. IF3 binds to the small subunit and prevents it from binding to the large subunit. The IF3 + small subunit complex binds to the ShineDalgarno sequence aligning it to the start site. IF2 binds to the charged tRNA and brings it to P site. IF1 then joins the small subunit complex by binding to the A site and blocks tRNAs from entering. This results in formation of the 30S initiation complex. Collectively IF1IF3 are called initiation factors. The IFs then disassociate from the complex and the large subunit can bind forming the 70S initiation complex. At this point the A, P and E sites are fully formed and the A site is unblocked, so that new tRNAs can enter. Elongation can begin. The change in conformation requires energy in the form of GTP, which is hydrolyzed resulting in GDP and Pi. In eukaryotes Similar with the following exceptions: The poly(A) tail with PAB proteins attached interacts with eIF4G and eIF4E bound to the 5’ cap (after the eIFs bind to the 5’ cap) and a circular structure forms. The 5’ cap recruits the small subunit (40S ribosome) plus initiation factors (including eIF3, eIF2, eIF4E/G and more). The complex scans 5’ to 3’ for the start codon (AUG), which is embedded in the sequence 5’ACCAUGG3’ (kozak sequence). Finding the start codon triggers changes which allow the large subunit to bind to the small subunit. 3. Elongation protein synthesis starts with Met (or fMet) bound to the P site. The next charged tRNA is brought into the A site (as are all subsequent tRNAs following initiation). Peptidyl Transferase Activity (thought to result from a ribozyme that is part of the large subunit) catalyzes the formation of a peptide bond. The tRNA in the P site is released (E site). Translocation occurs with the ribosome moving 5’ to 3’ to the next codon. The tRNA in the A site is now in the P site and a new tRNA can bind to the A site allowing the process of protein synthesis to continue. In prokaryotes EFTu, GTP and a charged tRNA form a complex that enters the A site. EFTs is involved in recycling the EFTu plus GTP complex. Translocation requires EFG and GTP for energy. Collectively EFTU, Ts and G are called elongation factors. In eukaryotes The process is similar with eEF1A instead of EFTu and eEF2 instead of EFG. 4. Termination stop synthesis when STOP codons (UGA, UAA, or UAG) are encountered. The polypeptide chain is cleaved from the last AAs tRNA and the ribosome detaches from the mRNA. Release factors are involved that bind to the stop codon (no AAs are coded for by stops). In prokaryotesRF1 recognizes UAA or UAG, RF2 recognizes UGA or UAA. RF3 binds GTP and the ribosome resulting in cleavage of the last AA in the chain from the tRNA in the P site, release of the tRNA and the mRNA, and disassociation of the ribosome. Collectively these are known as release factors. In eukaryotes, the process is similar. Study Guide Questions: The basic structure of an amino acid. Contain a central carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and a variable R group that is different for each amino acid The basic structure of a polypeptide. Polypeptides are made of amino acids linked by peptide bonds. All polypeptides have an amino terminus and carboxyl terminus. Synthesis begins at the amino terminal (Nterminus)/(NH )2and ends at the carboxyl terminal (Cterminus)/(COOH). The genetic code: how many possible codons, how many standard AAs, how many bases in a codon etc. There are 64 codons (3 are stop/nonsense codons and 61 are sense codons that code for amino acids), 20 standard amino acids, and three bases in a codon. There are more codons than anticodons. Know what ‘wobble’ is, and what property of the genetic code it is related to. Wobble is the flexibility of the third base of a codon, allowing different codons to code for the same amino acid. It is related to the degenerate property of the genetic code. Know the types of R groups (positive charge, negative charge, hydrophobic and hydrophilic) and their basic properties (you don’t need to memorize all the amino acids that are polar etc.). o Hydrophobic (nonpolar): no charge, no Hbonds, repel water o Hydrophilic (polar): slight charge, Hbonds, attract water o Positive charge (+): electrically charged at pH 7.0, can form hydrogen bonds and ionic bonds o Negative charge (): electrically charged at pH 7.0, can form hydrogen bonds and ionic bonds Know how these can affect properties of the protein like overall charge and position of side chains within the tertiary structure. Polar hydrophilic groups are on the outside and nonpolar hydrophobic groups are on the inside Know examples of how proteins can be posttranslationally modified. N and C terminus amino acids are removed or modified (e.g., removal of Nterminus methionine). Methyl groups or phosphates are added to particular amino acids or carbohydrate side chains may be attached or metals added to the protein (e.g., phosphorylation of serine). Polypeptides may be shortened, signal sequences removed or disulfide bonds may be added to connect peptide chains (e.g., processing of insulin). Know what primary, secondary, tertiary and quaternary protein structures are. o Primary: sequence of amino acids linked by peptide bonds in a polypeptide o Secondary: structure made of peptide backbone, stabilized by hydrogen bonds between groups (configuration in space/interactions between amino acids); includes alpha helixes and beta pleated sheets. o Tertiary structure: conformationoverall shape, three dimensional configuration that is specific to a given protein; structure made up of bonds or other interactions between R groups or between R groups and the peptide backbone; folding can be spontaneous or be aided by protein chaperones o Quaternary structure: association of multiple peptides to form a functional protein and applies only to proteins composed of more than one polypeptide chain; proteins with this structure are called oligomeric proteins Know what alpha helixes and beta sheets are and be able to identify them in a figure showing tertiary structure. o Alpha helix: a secondary structure of proteins characterized by a single, spiral chain of amino acids stabilized by hydrogen bonds; righthanded helix with 3.6 amino acids per turn; can form coils of coils; side chains stick outward o Beta pleated sheets: secondary structure in proteins that consists of two or more parallel adjacent polypeptide chains arranged in such a way that hydrogen bonds can form between the chains; can be parallel or antiparallel; side chains alternate sticking up or down; hydrogen bonds between the NH and CO groups hold the sheet together The following diagram illustrates a step in the process of translation. Identify the following elements on the diagram. o a) 5’ and 3’ end of the mRNA *above* o b) A, P and E sites in the ribosome *above* o c) Amino and carboxyl ends of the newly synthesized polypeptide chain *above* o d) A covalent bond that will be cleaved *above* o e) Where the next peptide bond will form *above* o f) What enzymatic activity is required for the peptide bond to form? Peptidyl transferase activity o g) What is the next step in translation after the peptide bond forms and what kind of proteins are required for this to happen? Termination; release factors 1, 2, and 3 are required for termination of translation to occur Given a diagram of a tRNA, label the anticodon loop and the AA acceptor site. Arrange the following components of translation in the order in which they would appear or be used in protein synthesis: 70S complex, release factors, peptidyl fMet fMet transferase activity, 30S subunit, and fMettRNA . 30S subunit fMettRNA 70S complex peptidyl transferase activityrelease factors Explain how initiation of translation differs between prokaryotes and eukaryotes. o Prokaryotic initiation: Initiation factors 3, 2, and 1 are involved in prokaryotic initiation. IF3 binds to the small subunit and prevents it from binding to the large subunit. The IF3 + small subunit complex binds to the ShineDalgarno sequence aligning it to the start site. IF2 binds to the charged tRNA and brings it to P site. IF1 then joins the small subunit complex by binding to the A site and blocks tRNAs from entering. This results in formation of the 30S initiation complex. Collectively IF1IF3 are called initiation factors. The IFs then disassociate from the complex and the large subunit can bind forming the 70S initiation complex. o Eukaryotic initiation: Similar to prokaryotic initiation with the following exceptions: The poly(A) tail with PAB proteins attached interacts with eIF4G and eIF4E bound to the 5’ cap (after the eIFs bind to the 5’ cap) and a circular structure forms. The 5’ cap recruits the small subunit (40S ribosome) plus initiation factors (including eIF3, eIF2, eIF4E/G and more). The complex scans 5’ to 3’ for the start codon (AUG), which is embedded in the sequence 5’ ACCAUGG3’ (kozak sequence). Finding the start codon triggers changes which allow the large subunit to bind to the small subunit. Explain the function of the A, P, and E sites during translation. o Aminoacyl/acetyl “acceptor” (A) site: one of the three sites in a ribosome occupied by a tRNA in translation; all charged tRNAs (with the exception of the initiator tRNA) first enter the A site in translation o Peptidyl “polypeptide” (P) site: in the elongation state of protein synthesis, tRNAs move from the aminoacyl (A) site into the P site o Exit (E) site: in the elongation stage of translation, the tRNA moves from the P site to the E site from which it then exits the ribosome What would happen during [transcription or translation] if the kozak sequence was deleted (for a eukaryotic gene)? Translation would not be able to be Chapter 12: Gene Expression Regulation Definitions: 1. Gene regulation: mechanisms and processes that control the phenotypic expression of genes 2. Regulatory gene: DNA sequence that encodes a protein or RNA molecule that interacts with DNA sequences and affects their transcription or translation or both 3. Constitutive gene (constitutively expressed): a gene that is not regulated and is expressed continually 4. Regulatory element: DNA sequence that affects the transcription of other DNA sequences to which it is physically linked 5. Operon: set of structural genes in a bacterial cell along with a common promoter and other sequences (such as an operator) that control the transcription of the structural genes 6. Operator: DNA sequence in the operon of a bacterial cell; a regulator protein binds to the operator and affects the rate of transcription of structural genes 7. Negative control: gene regulation in which the binding of a regulatory protein (repressor) to DNA inhibits transcription 8. Positive control: gene regulation in which the binding of a regulatory protein (activator) to DNA stimulates transcription 9. Inducible operon: operon in which transcription is normally off; something must happen for transcription to be induced, or turned on 10. Repressible operon: operon in which transcription is normally on; something must take place for transcription to be repressed, or turned off 11. Inducer: substance that stimulates transcription in an inducible system of gene regulation; usually a small molecule that binds to a repressor protein and alters that repressor so that it can no longer bind tot DNA and inhibit transcription 12. Allosteric protein: 13. Transcriptional activator protein: protein in eukaryotic cells that binds to consensus sequences in regulatory promoters or enhancers and affects transcription initiation by stimulating or stabilizing the assembly of the basal transcription apparatus 14. Coactivator: protein that cooperates with an activator of transcription; coactivators often physically interact with transcriptional activators and the basal transcription apparatus 15. Enhancer: SEQUENCE that STIMULATES maximal transcription of DISTANT genes; affects only genes on the same DNA molecule, contains short consensus sequences, is not fixed in relation to the transcription start site, can stimulate almost any promoter in its vicinity, and may be upstream or downstream of a gene; the function of an enhancer is independent of sequence orientation 16. Silencer: SEQUENCE that has many of the properties possessed by an enhancer but REPRESSES transcription of DISTANT genes 17. Insulator: DNA sequence that blocks or insulates the effect of an enhancer; must be located between the enhancer and the promoter to have blocking activity; also may limit the spread of changes in chromatin structure 18. Response elements (TF binding sites): Study Guide Questions: Enhancers, Silencers and Insulators in regulatory regions (what do they do at a basic level). Enhancers: distant sequences to which activators bind and stimulate transcription and are capable of affecting transcription at distant promoters Silencers: distant sequences to which repressors bind to inhibit transcription; unlike repressors in bacteria, most eukaryotic repressors do not directly block RNA polymerase Insulators: aka boundary elements, DNA sequences that block or insulate the effect of enhancers in a positiondependent manner; it blocks the action of the enhancer if it lies between the enhancer and the promoter; it has no effect if it lies outside the region between the enhancer and the promoter Know the basic differences between prokaryotic and eukaryotic regulation and how they are related to gene structure differences. o Prokaryotic mRNA is not processed before translation o Translation of prokaryotic mRNA can begin before transcription is complete o Transcription is the key step at which PROKARYOTES regulate gene expression o Prokaryotes have no introns o Prokaryotic transcribed regions contain multiple genes called polycistronic RNA o Prokaryotic genes are organized into operons o A regulatory region called an operator is between the promoter and the coding region o Different types of regulators can change how genes are expressed by binding to the operator; the operator affects expression of multiple genes Know the difference between genes (in prokaryotes) that are classified as negative inducible, negative repressible, positive inducible, positive repressible. Note negative means the regulator is a repressor, positive means it is an activator, inducible means the default state is “OFF” and repressible means the default state is “ON”. Know the levels at which a gene’s expression can be regulated and mechanisms for regulation at each level. o Transcription: number of premRNA molecules, transcriptional regulation o RNA processing: RNA stability, post transcriptional regulation, type of protein that is made (alternative splicing) o Translation: how much protein is made o Posttranslation modification: post translational regulation, activity of the protein Know how gene expression is affected by different types of transcriptional, posttranscriptional (after transcription or at translation, before translation is complete) and posttranslational regulation (after translation). o Transcriptional: on at some rate/off; subject to influence by internal or external cellular environment; major point of control of expression in development and in response to external changes in the environment (e.g. presence of allergens!) o Posttranscriptional: message stability/degradation; cap and tail additionschanges the amount of time an mRNA can be used and involves mechanisms for getting rid of message mistakes; can involve RNAi regulation and RNA splicing (in nucleus); RNA localization o Translational: control where/when the mRNA is used to make protein; the major point of control is initiation (this is an easy step at which translation can be blocked) o Posttranslational: modifications made to the protein product (lipid, sugar, methyl, acetyl, phosphate); protein stability; protein activity/conformation; determines the localization and the function of the protein in the cell Given a diagram of a simple case of constitutive, inducible or repressible regulation determine if a prokaryotic gene will be turned on or off. *below is positive repressible* Answer conceptual questions
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