Download Section 8: Genetic Mutations, Ribosome Structure

Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline Section 8: Genetic Mutations, Ribosome Structure,
and Tetracycline
TOPICS
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Ribosome structure
Classes of mutation
Mechanism of action of the tetracyclines
SUMMARY
Up to this point, our focus has been on antibiotics that exert prokaryotic specificity by
targeting molecules that are not present in eukaryotes (or in the case of gramicidin, no
robust prokaryotic specificity is exerted). The RNA polymerase and protein synthesis
inhibitors highlight examples of antibiotics that target prokaryotic-specific regions of
conserved molecules present in both prokaryotes and eukaryotes. We primed students for
the upcoming discussion upon introduction of trimethoprim. Now we elaborate on the
effect of mutations on structure. To understand the mechanism of action of the
translational inhibitors, such as the tetracycline class of antibiotics, students must
understand the consequences of mutations on RNA, how these mutations can affect
amino acid composition, the effects of those changes on structure and how structure
relates to function. We show that even subtle differences in structure between prokaryote
and eukaryote versions of the ribosome can result in altered binding affinity of antibiotic
for target. While we do not specifically discuss the transcriptional inhibitor rifampicin, it
too targets a prokaryotic region of a conserved molecule (RNA polymerase).
LEARNING GOALS
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Explain the molecular mechanism by which translational inhibitors kill bacteria.
Indicate the key molecules that interact with the ribosome and the function of
each interaction.
Provide a molecular explanation for how the transcriptional and translational
inhibitors achieve prokaryotic specificity.
Compare a silent, nonsense and missense mutation in terms of DNA alteration,
result on mRNA structure and polypeptide structure and function.
Explain how point mutations in a gene might result in a functional, yet
structurally altered gene product.
PRE-CLASS PREPARATION
Students should read about or review the mechanistic process of translation, classes of
mutation, DNA replication, and nucleic acid synthesis.
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline PRE-CLASS ASSESSMENT
1. During nucleic acid synthesis, the new, incoming nucleotide is always added to the:
A. 5’ phosphate
B. 5’ –OH
C. 3’ phosphate
D. 3’ –OH
Nucleic acids are always synthesized in the 5’ to 3’ direction.
2. Which of the following mutations would be MOST likely to have a harmful effect on
an organism?
A. A base-pair substitution in the middle of the coding sequence.
B. A deletion of three nucleotides in the middle of the coding sequence.
C. A single nucleotide deletion in the middle of an intron.
D. A single nucleotide deletion near the end of the coding sequence.
E. A single nucleotide insertion just after the start of the coding sequence.
Of course we can only determine the answer empirically, but we can speculate
based on knowledge of information flow in the cell. In all cases, we are assuming
that a polypeptide is the gene product. One might ask the students if and how their
answers would change if the gene product were an RNA.
A. A single base-pair substitution would keep the reading frame in tact, so
the mutation would likely be less harmful than a mutation that completely
alters the reading frame or truncates or eliminates the polypeptide.
B. While more nucleotides are removed than in (A), this change would
also keep the reading frame intact.
C. The introns don’t contain coding sequence, so this would not be likely
to have an adverse effect unless the sequence was regulatory in nature.
D. This change would cause a frameshift, but since it occurs at the end of
the coding region, the effect is probably less severe than the deletion in the
middle of the coding sequence (fewer amino acids affected).
E. This is likely to be the most severe because it would alter the reading
frame of the entire protein, resulting in the production of all the wrong
amino acids.
3. Choose the correct statement:
A. Mutations in the DNA are sometimes beneficial to the individual and the
population.
B. Mutations in the DNA are sometimes beneficial to the individual, but always harmful
to the population.
C. Mutations in the DNA are always harmful to the individual but sometimes beneficial
to the population.
D. Mutations in the DNA are always harmful to both the individual and the population.
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline This question assesses students understanding of the fact that mutations are not
always harmful and that they drive evolution. Students may not be able to answer
this question correctly prior to class, but if an unacceptable number of students
answers incorrectly, instructors can remind students of the question prior to class
and indicate that it will be asked again at the end of class.
GUIDE TO THE POWERPOINT SLIDES
Outline
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Introduction/active Learning
Ribosome structure
Tetracycline binding and prokaryotic specificity
Acquisition of genetic mutations
Classes of genetic mutations
Effect of mutation on structure and function
Review of transcription vs. translation
Introduction/active Learning
GUIDING QUESTIONS
• What interactions might be blocked by tetracycline interacting with its target?
• Is the target molecule present in prokaryotes and eukaryotes?
Active Learning
Activity type: Shout-out
Tetracycline kills bacteria by inhibiting translation. Students are shown a schematic
figure of the ribosome and other molecules used in translation with the following
question:
•
What interactions might be blocked by tetracycline interacting with its target?
This question is designed to encourage
students to consider the mechanism of
translation in greater detail than they
have up to this point by asking what
inter-molecular interactions are critical
the process and, therefore, would be
targets for disruption by tetracycline.
Consider?what?interac=ons?might?be?blocked?by?
tetracycline?interac=ng?with?its?target?
to
h"p://upload.wikimedia.org/wikipedia/commons/b/b1/Ribosome_mRNA_transla=on_en.svg?
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline There are a variety of different answers that are possible. Some potential answers include:
• mRNA interacts with (binds to) ribosome (30S subunit-5’ mRNA recognition)
• 30S interacts with AUG start site of mRNA
• 30S and 50S interact
• tRNA interacts with 70S ribosome (A site = aminoacyl tRNA entry site)
• Anticodon of tRNA binds to codon of mRNA
• Peptide bond formation catalyzes in the active site of the ribosome (=P site
peptidyltransferase)
• Spent (uncharged) tRNAs dissociate/exit (E site)
•  Tetracycline;binds;to;the;30S;(small);subunit;nucleoBdes;
through;nonCcovalent;bonding;interacBons;
• Growing polypeptide chain needs to be released
•  The;binding;alters;the;structure;of;the;small;subunit;such;that;;
the;tRNA;anBcodon;and;mRNA;codon;interacBons;are;
(exit tunnel)
obscured;
Actual answer: Tetracycline binds to the 30S (small)
subunit nucleotides through non-covalent bonding
interactions
The binding alters the structure of the small subunit
such that the tRNA anticodon and mRNA codon
interactions are obscured
h"p://faculty.ccbcmd.edu/courses/bio141/
lecguide/unit2/control/tetres.html;
Ribosome structure
We provide structural slides from the literature to
illustrate the tertiary structure of ribosomes as it
pertains to function, and the very specific noncovalent binding interactions required to achieve
tetracycline binding. The goal is for students to
connect how molecular structure determines
conformation and function.
This%figure%shows%the%general%shape%of%the%whole%ribosome%
Just as polypeptides use non-covalent bonding
interactions to adopt functional folded structure (conformation), RNA molecules can also
exert intra-molecular non-covalent bonding, primarily in the form of hydrogen bonds
between complementary base pairs.
The rRNA associates with the small polypeptides through non-covalent bonding
interactions. This “folded” structure of the rRNA plus polypeptides confers the shape and
function of the molecule. The large and small subunit associate only in the presence of
mRNA which passes through a “tunnel” created by the mature ribosome.
If:we:zoom:in:on:the:RNA:porBon,:we:see:that:
individual:nucleoBdes:undergo:baseFpairing:to:form:
“stems”:and:“loops”:
•  The$large$and$small$subunit$associate$only$in$the$presence$of$mRNA$
•  The$mRNA$passes$through$a$“tunnel”$created$by$the$mature$ribosome$
•  This$tunnel$contains$the$ac;ve$A,$P,$and$E$sites$where$charged$tRNA$
molecules$enter$(A$site),$amino$acids$are$transferred$to$the$growing$
chain$(P$site)$and$uncharged$tRNAs$exit$(E$site)$
Protein$synthesis$inhibitor$
an;bio;cs,$such$as$tetracycline,$
bind$to$the$30S$or$50S$
ribosomal$subunit.$$
Where$on$these$subunits$do$
you$think$they$bind?$
h"p://rna.ucsc.edu/rnacenter/images/figs/ecoli_23s.jpg:
Small World Initiative ICell
nstructor Guide 1146
Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline This tunnel contains the active A, P, and E sites where charged tRNA molecules enter (A
site), amino acids are transferred to the growing chain (P site) and uncharged tRNAs exit
(E site).
ell
146
&
The&primary&binding&site&of&tetracycline&(yellow)&is&to&
the&A&site&of&the&ribosome&
Green,&blue&and&cyan&represent&
relevant&helices&of&the&16S&rRNA&
Model&depic*ng&possible&tetracycline&binding&
interac*ons&with&16S&rRNA&&
Cell
1146
The&point&here&is&not&to&memorize&this&specific&example,&but&to&use&it&as&
context&to&understand:&
•  The&role&of&nonDcovalent&bonding&interac*ons&in&biology&
•  The&rela*onship&of&structure&to&func*on&
•  How&the&ribosome&func*ons&at&the&molecular&level&
tetracycline&
From&Broderson&(2000)&Cell&103:1143N&1154&
h<p://commons.wikimedia.org/wiki/
File:Tetracycline_structure.svg&
tetracycline&
Broderson&(2000)&Cell&103:1143D&1154&
At this point, students should be able to meet the learning goal:
ü Indicate the key molecules that interact with the ribosome and the function of
each interaction.
Tetracycline binding and prokaryotic specificity
GUIDING QUESTION:
• How does tetracycline achieve prokaryotic specificity if it targets a molecule
conserved in both prokaryotes and eukaryotes?
Transcription, translation and DNA synthesis are highly conserved processes, present
across phylogeny. We ask, how then, do antibiotics that target these functions achieve
prokaryotic specificity? The answer lies in the fact that there are slight differences in the
structure of the targets; however, while there are structural differences between
prokaryotic and eukaryotic targets, function of the target molecule is retained.
We begin by presenting the DNA differences between the prokaryotic and eukaryotic
ribosome sequences that alter ribosome structure to the extent that tetracycline binding
specificity is affected without affecting function. These special DNA mutations are what
allow utility of antibiotics that target functions that are conserved in both eukaryotes and
prokaryotes. The transcriptional inhibitor rifampicin utilizes this strategy as well,
although we do not include slides depicting its mechanism of action. Note that the
original paper for discussion listed in Section 7 (Fujii et al. (1995) provides an
opportunity to discuss the mechanism of action of rifampicin.
Active Learning
Activity type: Think-Pair-Share
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline We use an example from the literature to illustrate that tetracycline achieves its
prokaryotic specificity due to a higher binding affinity for the prokaryotic ribosome
structure.
Interpret'this'graph''
Question: Interpret this graph (see image on
slide)
[3H]Tet'refers'to'
tetracycline'in'which'
hydrogen'is'replaced'
with'the'radioac9ve'
[3H]'form.''
How#does#the#informa,on#gained#in#this#experiment#differ#
from#that#in#the#previous#experiment?#
In#vitro#
transla,on#with#
purified#
ribosomes#and#
poly#U#RNA#
template#
100%#
corresponds#to#
number#of#phe#
amino#acids#
incorporated#
with#no#
tetracycline#
present.##
Cell
1146
V1*
V2*
V3*
V4*
V5*
V6*
V7*
1243*
1294*
Cell
1146
1117*
Tetracycline*binding*
1054* 1197D1198*
Numbers*refer*to*single*
nucleo3de*posi3ons*in*the*
16S*rRNA*
986*
The$process$of$transla/on$is$conserved$between$prokaryotes$
and$eukaryotes;$key$nucleo/de$differences$in$ribosomal$RNA$
retain$func/on$of$the$ribosome,$but$create$differen/al$
binding$opportuni/es$for$an/bio/cs$
Budkevich T V et al. Nucl. Acids Res. 2008;36:4736-4744
1173*
The second slide measures ribosome function in the
presence of tetracycline. An in vitro translation assay
was performed using a polyU RNA template
together with purified ribosomes to produce a polyphenylalanine peptide (codon UUU). Prokaryotic
ribosome function decreases markedly in the
presence of tetracycline.
Budkevich T V et al. Nucl. Acids Res. 2008;36:4736-4744
1043*
An in vitro binding assay was performed
between a radioactive ([3H]) form of tetracycline
and either purified prokaryotic (70S) or
eukaryotic (80S) ribosomes. The data show
higher levels of tetracycline bound to the
prokaryotic (70S) ribosome compared to the
amount of tetracycline bound to the eukaryotic (80S)
ribosome.
V8*
V9* V10*
tetracycline*
Broderson*(2000)*Cell*103:1143D*1154*
Lafontaine$and$Tollervey$(2001)Nature)Reviews)Molecular)Cell)Biology)2,$514H520$
In the slide set, we elaborate on nucleotide
differences between the eukaryotic and
prokaryotic rRNA sequences. These
nucleotide differences are unique in that they
lead to slightly different structures without
altering overall functional capability. Even
subtle differences in structure can result in
altered binding affinity of antibiotic for target.
Transcrip)onal,inhibitors,exploit,subtle,
differences,in,structure,between,prokaryo)c,
and,eukaryo)c,RNA,polymerase,structure,,
Image,from,Centre,d’études,d’agents,Pathogènes,et,Biotechnologies,pour,la,Santé,
Montpellier*+*France,,
h<p://www.cpbs.cnrs.fr/spip.php?ar)cle81&lang=fr,
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline This information also becomes relevant for our discussion of molecular phylogeny in
Section 9 and will be relevant to students when they characterize the identity of their
isolates using 16S PCR amplification, sequencing and BLAST comparisons.
At this point, students should be able to meet the learning goal:
ü Provide a molecular explanation for how the transcriptional and translational
inhibitors achieve prokaryotic specificity.
Acquisition of genetic mutations
GUIDING QUESTION:
• How do these nucleotide changes arise and why are they maintained?
Presentation of ribosomal structural detail provides an excellent segue into the topic of
nucleotide changes and their potential effect on conformation and function.
DNA$polymerase$makes$an$occasional$
mistake$
3’
C 5’
G
T G
CT AAC
5’ GTGAAT GG C
3’ CACTTACC
G T
AA G
C C
G 3’
5’
Briefly, we present slides depicting how
mutations in DNA replication are
transmitted to subsequent generations of
cells and how these mutations can affect
polypeptide structure after translation.
For example, a single nucleotide change
can result in serine substitution in place
an alanine. We do not discuss the
mechanism of DNA replication; for those
wishing to do so, this would be an idea
placement of that topic.
A"serine"residue"is"subs+tuted"for"alanine"
DNA"
5’ ATG GCT TGC 3’
3’ TAC CGA ACG 5’
transcrip+on"
5’ AUGGCUUGC 3’
transla+on"
N Met–Ala–Cys C
5’ ATG TCT TGC 3’
3’ TAC AGA ACG 5’
transcrip+on"
5’ AUGUCUUGC 3’
transla+on"
N Met–Ser–Cys C
•  Are)the)proper7es)of)alanine)and)serine)similar?))
•  Do)you)think)that)this)single)amino)acid)subs7tu7on)will)
affect)the)conforma7on)of)the)polypep7de?)
•  If)so,)how?)If)not,)why?)
•  If)conforma7on)is)altered,)will)func7on)be)altered?)
of
Alanine)
Serine)
WikiCommons)user:)Borb)
Classes of genetic mutations
We provide schematic slides to illustrate the different classes of mutation: point
mutations, deletions, and insertions. We also discuss the different classes of point
mutations (normal, silent, nonsense, and missense) and their potential outcomes. It is also
important to stress the key concept that point mutations drive evolution.
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline We can use a three-letter word analogy to consider the
effects of some DNA mutations on amino acid sequence
HIT THE BAT
Normal “polypeptide” sequence
HIT CTH EBA T
Single nucleotide insertion
RESULT: “frameshift” of codon usage
Classes&of&point&muta/ons&
NORMAL&
SILENT&
NONSENSE&
MISSENSE&
TGC
ACG
TGT
ACA
TGA
ACT
TGG
ACC
transcrip/on&
UGC
transla/on&
HIT CAT THE BAT
Three nucleotide insertion
RESULT: single amino acid inserted; codon
usage remains “in frame”
HIT CHE BAT
Single nucletide substitution (point
mutation)
RESULT: silent, missense or nonsense
(see next slide)
Cys
Correct&
amino&acid&
transcrip/on&
UGU
transla/on&
Cys
No&change&
in&amino&
acid&
sequence&
transcrip/on&
transcrip/on&
UGA
UGG
transla/on&
transla/on&
STOP
Premature&
STOP&codon&
introduced&
Trp
Single&amino&
acid&change&
Effect of mutation on structure and function
Examina'on)of)Red)Blood)Cells)
)Sickle)Cell)Anemia))
)Normal))
)
2012)Northeast)Regional)Summer)Ins'tute))
)
)
We have inserted a modified version of a
“teachable tidbit” from the National Academies
Northeast Regional Summer Institute of 2012 to
demonstrate the effects of a single amino acid
change on hemoglobin in the disease sickle cell
anemia. While this deviates from our theme of
antibiotics, it is a well-studied example that
students usually find interesting. For an additional
example involving resistance to the antibiotic
ciprofloxacin, refer to the discussion paper packet
Number 7 (also found at the end of Section 10).
More information on the sickle cell anemia “tidbit” can be found at the Yale Center for
Scientific Teaching website:
http://cst.yale.edu/biology-and-chemistry-interface-tidbits
Active Learning
Activity type: Think-Pair-Share and electronic response
Think-Pair-Share:
In the first activity, students use their knowledge
regarding polarity to determine which amino acid side
chains are hydrophilic and which are hydrophobic.
ThinkEPairEShare(
1.(Which(amino(acid(side(chains((R(groups)(are(hydrophobic?(
2.(Which(amino(acid(side(chains((R(groups)(are(hydrophilic?(
Valine((Val,(V)((
Glutamate((Glu,(E)((
Serine((Ser,(S)(
Leucine((Leu,(L)(
Lysine((Lys,(K)((
Threonine((Thr,(T)(
2012(Northeast(Regional(Summer(InsJtute((
(
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline We then ask students to consider:
What percentage of the 574 amino acids in
hemoglobin would have to be changed to
cause sickle cell anemia?
Even if students are familiar with this
example, it is dramatic to consider (or
reconsider) the large implications for such a
seemingly small change. Note that there are
two β subunits that compose the mature
hemoglobin molecule, so there are actually
two amino acid changes per hemoglobin.
Each%blue%
dot=one%amino%
acid%
α=141%amino%acids%
%
β=146%amino%acids%
β%
α%
α%
β%
Approximately%what%percentage%of%the%574%amino%acids%in%hemoglobin%
do%you%think%you%would%need%to%change%to%cause%sickle%cell%anemia?%
2012%Northeast%Regional%Summer%InsItute%%
%
Electronic Response:
Next, analogous to the question posed previously about where tetracycline might be
expected to bind to the ribosome, we ask:
Where in this folded protein might this change have occurred?
A. Near the oxygen binding site
B. At the interface between subunits
C. On the surface
Remember:(An(external(hydrophilic(amino(acid(is(
replaced(with(a(hydrophobic(one((
If the comparison were totally analogous to the
tetracycline/ribosome example, we might expect
the amino acid change to occur where the
different subunits interact, but in this case, the
change is on the surface on the molecule.
Because a hydrophilic amino acid is exchanged
for a hydrophobic one, this region of the surface
no longer interacts with water and preferentially
interacts with other altered hemoglobin
molecules. We indicate to the students that the
answer must be determined through experimental
analyses and that all answers would be logical predictions.
Glutamate(
(hydrophilic)(
Valine(
(hydrophobic)(
2012(Northeast(Regional(Summer(InsCtute((
(
At this point, students should be able to meet the learning goals:
ü Explain the molecular mechanism by which transcriptional and translational
inhibitors kill bacteria.
ü Compare a silent, nonsense, and missense mutation in terms of DNA alteration,
result on mRNA structure, and result on polypeptide structure and function.
ü Explain how point mutations in a gene might result in a functional, yet
structurally different gene product.
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline Review transcription vs. translation
Active Learning
Activity type: Think-Pair-Share
We close with an activity designed to highlight the effects of mutation in RNA synthesis
compared to DNA synthesis and the implications of DNA replication errors for
prokaryotes compared to eukaryotes.
Question: Consider the effects of a nucleotide synthesis error that results in complete loss
of protein function and that the protein is required for life of the cell.
• First, consider the effect on an individual prokaryotic cell if the mutation was
introduced by RNA polymerase in transcription.
• Next, consider the effect on an individual prokaryotic cell if the mutation was
introduced by DNA polymerase in DNA replication.
Students tend to think of RNA synthesis as a single reaction, yet transcription occurs
continuously from a single gene, such that the effects of a single mutation in RNA
synthesis are diluted.
We use a visual representation to highlight this fact. On the other hand, an error in DNA
replication will affect all gene products in a bacterial cell (and half of those in a
eukaryotic cell since there are two copies of each gene).
With%an%error%in%DNA%replica1on%
Compare(the(effects(of(an(error(in(transcrip1on.(.(.(
Modified from Figure 7-2 Essential Cell Biology (© Garland Science 2010)
Modified from Figure 7-2 Essential Cell Biology (© Garland Science 2010)
In addition, mutations in RNA are not heritable. Errors in DNA synthesis, on the other
hand have dire consequences for the cell. In multicellular organisms, defects must occur
in germline (sex) cells to be heritable to the next generation (but the error can be
propagated to subsequent daughter cells, leading to defects in development or cancer).
Mammals have two copies of each chromosome (diploid), and only one is acquired by
each gamete. Therefore, a particular mutation in a eukaryotic cell may not be inherited.
All DNA mutations in bacteria are heritable; they only have one chromosome and each
cell division creates offspring.
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline Summary Statement:
Antibiotics must target essential (conserved) functions, yet they must be prokaryotespecific to be most useful.
Specificity is achieved in two main ways:
• Antibiotic targets a molecule that is essential for prokaryotes but not for
eukaryotes.
• Antibiotic targets a structural change in a conserved molecule. The structural
difference is the result of nucleotide differences between prokaryote and
eukaryote DNA that alters structure of the gene product but retains function.
POST-CLASS ASSESSMENT
1. What is the most likely effect of a mutation in the DNA that inserts a three nucleotide
stop codon into the beginning of the coding sequence of a gene?
A. The polypeptide and the mRNA will be shorter than usual.
B. The polypeptide will be shorter than usual but the mRNA will not be affected.
C. The polypeptide will be unaffected, but the mRNA will be shorter than usual.
D. In eukaryotes, only the polypeptide will be affected, but in prokaryotes the mRNA
may also be affected if several genes are under the control of the same promoter.
The transcript, in general, is unaffected by codon changes.
2. Consider a bacterial operon that contains the coding sequence for three genes, (A, B,
C), all under the control of a single promoter where A is the most upstream gene
followed by B then C. How will a three nucleotide insertion in the most conserved region
of the promoter most likely affect expression of gene C?
A. Protein C will probably not be produced.
B. The gene C mRNA will be produced but Protein C will be non-functional.
C. The gene C mRNA will be produced but Protein C will not be produced.
D. The expression of gene C will not be affected.
Because the genes are arranged in an operon, they are all under control of the
same promoter. Insertion of three nucleotides at the conserved region of the
promoter will likely negatively affect RNA polymerase holoenzyme binding,
leading to loss of expression of the entire operon (no mRNA, no protein).
Small World Initiative Instructor Guide Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline ORIGINAL RESEARCH PAPERS FOR DISCUSSION
The set of papers below give students a glimpse into the incremental nature in which
scientific models are tested and functions are elucidated. We illustrate the process of
discovery of the mechanism of action of tetracycline. We suggest that instructors focus
on one or a few key figures from each paper. The first paper in the series is from 1953
(the same year the structure of DNA was published) demonstrating that aureomycin (the
first tetracycline identified) affects protein synthesis. The second paper demonstrates
binding to the 30S subunit of the ribosome while the third paper shows binding to
ribosomes in a cell-free system to inhibit protein synthesis. The fourth and fifth papers
work to elucidate the specific region to which tetracycline binds. The final paper
determines specific tetracycline binding sites using atomic structure data and predicts the
mechanism of action based on these data.
Gale, E. and Folkes, J. (1953) The Assimilation of Amino-Acids by Bacteria. Biochem.
53:493-498.
Connamacher, R. and Mandel, G. (1965) Binding of Tetracycline to the 30S Ribosomes
and to Polyuridylic Acid. Biochemical and Biophysical Research Communications 20:98103.
Day, L. (1966) Tetracycline Inhibition of Cell-Free Protein Synthesis. J. Bact. 91:19171923.
Gottesman, M. (1967) Reaction of Ribosome-bound Peptidyl Transfer Ribonucleic Acid
with Aminoacyl Transfer Ribonucleic Acid or Puromycin. J. Biol. Chem. 242:5564-5571.
Goldman, R., Hasan, T., Hall, C., Strycharz, W., and Cooperman, B. (1983)
Photoincorporation of Tetracycline Into Escherichia coli Ribosomes. Identification of the
Major Proteins Photolabeled by Native Tetracycline and Tetracycline Photoproducts and
Implications for the Inhbitory Action of Tetracycline on Protein Synthesis. Biochem.
22:359-368.
Brodersen, D., Clemons, W., Carter, A., Morgan-Warren, R., Wimberly, B., and
Ramakrishnan, V. (2000) The Structural Basis for the Action of the Antibiotics
Tetracycline, Pactamycin, and Hygromycin B on the 30S Ribosomal Subunit. Cell
103:1143-1154.
RESOURCES
Budkevich et al. (2008) Features of 80S mammalian ribosome and its subunits. Nucleic
Acids Res. 36:4736-4744.
Tetracycline binding to ribosome experiment.
Pray, L. (2008) DNA replication and causes of mutation. Nature Education 1:214.
DNA replication and causes of mutation.