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Small World Initiative Instructor Guide Section 6: Information Flow Section 6: Information Flow TOPICS Introduction to central dogma Nucleic acid structure Mechanism of transcription and translation SUMMARY We have spent the last several sections discussing molecular differences between the prokaryotes and eukaryotes that serve as targets for antibiotic binding. Here we refer back to initial discussions in Section 2 where we introduced the tree of life. Observable (phenotypic) differences between groups in the tree arise from accumulation of heritable changes in the DNA. To introduce the central dogma, we can ask why changes in DNA result in observable changes (perhaps providing a student plate as an example—why do the different isolates look different?) We focus on nucleic acid structure and the central dogma at its most basic level—the mechanism of transcription and translation to produce functional proteins (or RNA) from the genome,.This then serves as a springboard to launch a more in-depth discussion of information flow in the upcoming sections. The overall aim is to guide students to a sophisticated conceptual understanding that goes beyond the mechanistic process of transcription and translation to encompass the potential effect of point mutations on structure, the importance of structure on function, and regulatory aspects of gene expression. Thus, we strongly suggest using significant class time for exercises that require students to apply understanding of the mechanistic processes of transcription and translation such that they have a solid foundation on which to build in the upcoming sections. To this end, we have provided a large number of active learning exercises from which instructors may choose. LEARNING GOALS Know how information is converted from gene to gene product (process of transcription and translation). • Know structure and function of key molecules involved in transcription and translation. — Recognize structural features of nucleotides and nucleic acid polymers, and be able to distinguish RNA from DNA. — Explain the “antiparallel” structure of double-stranded nucleic acid and the molecular rationale for complementary base-pairing interactions. — Know the role during gene expression of DNA, promoter, RNA polymerase, sigma factor, mRNA, ribosome, tRNA, and rRNA. — Compare and contrast transcription and translation. By the end of Section 8 • Explain the molecular mechanism by which transcriptional inhibitors kill bacteria. • Explain the molecular mechanism by which translational inhibitors kill bacteria. • Small World Initiative Instructor Guide Section 6: Information Flow PRE-CLASS PREPARATION Prior to class, students should read about nucleotide structure, RNA and DNA polymer structure, and the mechanisms of transcription and translation. PRE-CLASS ASSESSMENT 1. Translation is the process of creating a polypeptide from a _______________ template? a) single-stranded DNA b) double-stranded DNA c) messenger RNA (mRNA) d) transfer RNA (tRNA) 2. Which of the following classes of RNA are essential for translation? Choose as many as apply. a) tRNA b) mRNA c) rRNA d) microRNA 3. The anticodon can be described as a) a sequence in the tRNA that is identical to the corresponding codon in the mRNA. b) a sequence in the tRNA that determines which amino acid is bound to the 3’ end of the tRNA. c) located at the extreme 3’ end of the tRNA. d) required for regulation of transcription. 4. During transcription, the strength of non-covalent bonding interactions determines whether the correct (complementary) nucleotide is inserted or not. True or false. Explain. True. The correct, complementary nucleotide will have a stronger binding affinity than other (incorrect) nucleotides and thus will be in the “bound” position longer than other nucleotides. This increases probability that the enzyme (RNA polymerase) will catalyze the synthesis reaction required to add this nucleotide to the growing polymer. Generally, incorrect nucleotides do not remain bound long enough for the reaction to proceed. 5. If the termination codon is missing, the mRNA will be extra long. True or False. Explain. This question confounds students, and is probably best addressed in class if deemed worthy by the instructor. It reinforces the physical and temporal separation of transcription and translation. The RNA polymerase responds to information (regulatory sequences) present in the DNA while the ribosome responds only to information present in the mRNA. Thus, “codons” are not recognized by the RNA polymerase. Transcription termination will proceed as usual. Small World Initiative Instructor Guide Section 6: Information Flow GUIDE TO THE POWERPOINT SLIDES Outline • • • • Central dogma/information flow Introduction to mechanism of transcription — Structural features of nucleotides and nucleic acid polymers — RNA vs. DNA structural differences Introduction to mechanism of translation Practice with gene expression Central dogma/information flow We now turn to the transcription and translational inhibitors, but we first want to ensure that students have a foundational understanding of what those processes entail. We will study gene expression and its regulation throughout the next three sections. We can begin by asking students to observe their patches or single colonies and indicate why they may look different (shiny, crusty, pigmented, clear, etc.). Active learning Activity type: Independent problem-solving We display two strains with an observable difference—one has purple color due to production of a pigment called violacein. We ask students to think about the underlying basis for the difference. Why$is$the$strain$on$the$le.$purple$while$the$ one$on$the$right$is$not?$ h4p://microbiologiabrasil.blogspot.com/2009/01/ janthinobacteriumsp.html$ Copyright$©$Gary$E.$Kaiser$ h4p://faculty.ccbcmd.edu/courses/bio141/labmanua/lab2/sainsol.html$ We propose two interpretations: 1. One strain has genes for pigment biosynthesis, the other doesn’t. 2. Both strains possess the genes for pigment biosynthesis but expression patterns differ. Students need not report back at this point. The goal is to stimulate interest and thought regarding this observable difference. Small World Initiative Instructor Guide Section 6: Information Flow Gene$Expression$ We will revisit this question in more depth in Section 8. For now, we are going to focus on the first and most straight-forward explanation, using that as context to introduce the central dogma—the transfer of the information in the DNA to RNA and from mRNA to protein. Genes encode information to make proteins (or RNA) with a particular function. Elimination of a gene can lead to loss of the gene product which is sometimes observable as a phenotype and is the case in the photo above. The gene encoding an enzyme necessary for pigment biosynthesis is missing in the strain on the right. A photo of a student patch plate with a diversity of observable phenotypes could be inserted to coax students to consider the interpretations for the active learning slide plates in the context of their own results. Process$by$which$informa3on$in$a$gene$is$ converted$into$func3onal$gene$product$ (usually$a$protein,$but$can$be$RNA)$ DNA$(gene)$ tRNA! transcrip3on$ rRNA! RNA$ tRNA$$$ microRNA$$ mRNA$$ rRNA$$$$$$$$$$$$ transla3on$ Polypep3de(s)$ transcrip8on" transla8on" Enzyme"gene" Enzyme"mRNA" Enzyme" transcrip)on+ transla)on+ Enzyme+gene+ Enzyme+mRNA+ Enzyme+ Gene"for"enzyme"missing:" • No"transcrip8on"of"enzyme" • No"transla8on"of"enzyme" • No"enzyme"protein" • No"purple"pigment"accumulates" Enzyme'#1' A+ Pigment+ precursor+ B+ Purple+ Pigment+ Genes'encode'the'instruc3ons'for'building'gene$products$ (usually'proteins'but'also'some'RNAs)'that'carry'out' regulatory,'enzyma3c'or'structural'roles'in'the'cell' mRNA' Purple'colony' Transla3on'' Transcrip3on'' Enzyme'gene' A" Pigment" precursor" B" No" pigment" We provide slides to visually explain the loss of gene and corresponding gene product for the active learning exercise. transla3on' transcrip3on' Enzyme'gene' Enzyme'#1' mRNA' White'colony' The'purple'colony'is'gene3cally'iden3cal'to'the'white'colony'except'the'white' colony'is'missing'one'gene.' Note: This is an ideal opportunity to introduce the operon organization of bacterial gene clusters. We revisit operons and provide schematic slides in Section 7 in the context of gene regulation. We also encourage instructors to insert slides to illustrate single gene defects in humans. We provide some examples to consider (cystic fibrosis, sickle-cell anemia (discussed in Section 8), color blindness, phenylketonuria). At this point, students should be able to meet the learning goals: ü Know how information is converted from gene to gene product (process of transcription and translation). There%are%many%human%condi/ons% resul/ng%from%a%single%gene%defect% • Cys/c%fibrosis—defect%in%gene%encoding%protein%that% transports%ions%across%epithelial%cell%membrane.% • Sickle%cell%anemia—defect%in%gene%encoding%hemoglobin;% hemoglobin%is%produced,%but%with%altered%conforma/on%due%to% single%amino%acid%change.% • Color%blindness—defect%in%single%gene%encoding%a% photopigment%in%the%re/na.% • Phenylketonuria—a%metabolic%disorder%resul/ng%in%intellectual% disability;%loss%of%gene%encoding%enzyme%that%catalyzes% conversion%of%phenylalanine%to%tyrosine.% % Small World Initiative Instructor Guide Section 6: Information Flow Introduction to mechanism of transcription Active learning Activity type: Shout out Question: What do you know about transcription? What key molecules are involved and what is their role? It is the expectation that students will have read about the mechanics of transcription prior to class, but we can engage everyone and identify and address possible misconceptions by asking the above question and collecting answers. It is helpful to record student responses electronically. Misconceptions may best be addressed immediately while others may be best addressed as instructors progress through the lectures. At this point, instructors may review the Transcrip)on+ mechanistic aspects of transcription and nucleotide/nucleic acid structure at the level of detail that they feel is appropriate for their classroom. In addition, instructors wishing to RNA+synthesis+ cover the mechanism of DNA replication may • Requires+DNA+as+a+template.+ • RNA+polymerase+binds+double;stranded+DNA,+causes+hydrogen+ want to do so here or in Section 8. Initiation of bonds+to+separate,+catalyzes+synthesis+of+RNA+nucleo)de+polymer+ complementary+to+template+DNA.+ transcription (e.g. promoters, • Occurs+5’+to+3’+(monomer+nucleo)des+are+added+to+the+3’+–OH).+ sigma/transcription factors, etc.) is covered in Section 7. At this point, students should be able to meet the learning goals: ü Recognize structural features of nucleotides and nucleic acid polymers, and be able to distinguish RNA from DNA. ü Explain the “antiparallel” structure of double-stranded nucleic acid and the molecular rationale for complementary base-pairing interactions. Introduction to mechanism of translation Active learning Activity type: Independent problem-solving Three different active learning options are presented. Each option takes a different approach to reinforce the mechanics of translation. All exercises assume that students have done reading on the topic prior to class. Small World Initiative Instructor Guide Ac6ve%learning%op6on%#1% What%do%you%know%about%the% molecular%mechanics%of%transla6on?% Section 6: Information Flow Ac7ve'learning'op7on'#2' Arrange'the'following'in'order:' A.' C.' What%molecules%are%involved?% In%the%schema6c%diagram,%label%each%of%the%following%molecules:% • • • • • • • mRNA% ribosomal%small%subunit% ribosomal%large%subunit% tRNA% amino%acid% codon% an6codon% D.' B.' E.' Active learning option #1 and 2 take a visual approach to the process. In the first, students label a schematic with appropriate molecule names. In the second, students order the process. Instructors may print out the figures prior to class; alternatively, the second could be converted to an electronic-response Ac've)learning)op'on)#3) question. Active learning option #3 stresses an understanding of the order of process. For this third option, instructors should print the text prior to class and cut the statements into strips that the students must place in the right order (or provide scissors and ask the students to do the cutting). Transla'on)strip)sequence)) • Ribosome)reaches)a)stop)codon,)release)factor)binds)to)A9site.) • Pep'de)bond)between)tRNA)and)polypep'de)is)hydrolyzed,) releasing)polypep'de.)) • Ribosome)small)subunit)binds)mRNA)at)a)ribosome)binding)site) • Ini'ator)charged)(aminoacyl)9tRNA)and)large)subunit)associate)with) the)ribosome/mRNA)complex.)) • Ribosome)small)subunit)“slides”)along)mRNA)un'l)transla'onal) start)(AUG))sequence)is)recognized.)) • Pep'de)bond)is)formed,)uncharged)ini'ator)tRNA)exits)ribosome.)) • Coding)amino)acids)enter)A9site,)pep'de)bonds)are)formed,)then) uncharged)tRNAs)exit)ribosome.) • New)charged)tRNAs)enter)the)A9site)of)the)complete)ribosome;) ini'ator)tRNA)occupies)P9site.)) Instructors should include content slides to the extent they feel necessary. We also suggest taking advantage of the many instructional videos available. Practice with gene expression Active learning Activity type: Independent problem-solving Transcribe$the$following$DNA:$ 3’ TAC CGA ACG 5’ DNA$ Here, we offer a very basic practice opportunity for students to engage in the gene expression mechanics. We start with this easy example, upon which we will build complexity in the upcoming sections. Ultimately, the goal is to impress upon students Small World Initiative Instructor Guide Section 6: Information Flow that regulatory sequences must be present to guide the transcription and translational factors to the correct sites along the nucleic acids. We suggest posing the question as indicated, allowing a minute or so for response, then posting the second slide to remind students that thymine is replaced by uracil. Transcribe$the$following$DNA:$ 3’ TAC CGA ACG 5’ DNA$ Remember$that$in$RNA,$,$thymine$ Often there will be groans (they knew, but had (T)$is$replaced$by$uracil$(U)$ forgotten). The translation activity is equally simple, so much so that students often acquire a false sense of security in their understanding of gene expression. With our final guiding question below, we begin to convey that there are nuances that students may not have considered. While beautifully elegant, gene expression is not the straight-forward process that many introductory-level students believe it to be. At this point, students should be able to meet the learning goals: ü Know the role during gene expression of DNA, RNA polymerase, mRNA, ribosome, tRNA, and rRNA. ü Compare and contrast transcription and translation. ü Know the structure and function of key molecules involved in transcription and translation. GUIDING QUESTION • How is the correct strand of the double-stranded DNA chosen as the template for transcription? Instructors may wish to set the stage for upcoming discussions by posting this question at the end of class Small World Initiative Instructor Guide Section 6: Information Flow POST-ASSESSMENT 1. The photo shows two different strains of bacteria that are identical except that the strain on the left produces an extracellular capsule composed of polysaccharide. Propose a hypothesis to explain the underlying molecular difference between the two strains. J. Exp. Med. 98:21, 1953. Based on materials in the lecture, students will likely propose that the strains differ due to the presence of a particular gene in one strain vs. the other. In this example, the strain on the left contains genes required to synthesize the capsule while absent in the strain on the right. An alternate hypothesis is that the organisms contain the same genes, but the strain on the right is grown under different conditions that trigger production of the capsule. 2. Would an antibiotic that targets RNA polymerase be expected to have the same effect on a bacterial cell as one that targets the ribosome? Explain. Both processes are critical for expression of all genes in the cell, so blocking either would be expected to have the same general outcome for the cell. RESOURCES Overview of transcription and translation excerpted from Essentials of Cell Biology, Unit 2.1 and presented in Scitable by Nature Education http://www.nature.com/scitable/topicpage/ribosomes-transcription-and-translation14120660 Organization of bacterial genes into operons Ralston (2008) Operons and Prokaryotic Gene Regulation Nature Education 1:216 Transcription in eukaryotes http://vcell.ndsu.nodak.edu/animations/transcription/movie-flash.htm Translation http://www.youtube.com/watch?v=5bLEDd-PSTQ