Download Section 6: Information Flow

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