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Sec$on 8 Gene$c Muta$ons, Ribosome Structure and the Tetracyclines Explain the mechanism of ac$on of the transcrip$onal and transla$onal inhibitors Requires knowledge of: Sec$on 6 Sec$on 7 Sec$on 8 •  How informa$on is converted from gene to gene product (process of transcrip$on and transla$on). •  Structure and func$on of molecules involved in transcrip$on. •  Structure and func$on of molecules involved in transla$on. •  Regula$on of transcrip$onal ini$a$on. •  Differences in gene expression between prokaryotes
and eukaryotes. •  Rela$onship of DNA muta$on to protein structure and func$on. Explain the mechanism of ac$on of the transcrip$onal and transla$onal inhibitors Requires knowledge of: Sec$on 6 Sec$on 7 Sec$on 8 ü How informa$on is converted from gene to gene product (process of transcrip$on and transla$on). ü Structure and func$on of molecules involved in transcrip$on. ü Structure and func$on of molecules involved in transla$on. ü Regula$on of transcrip$onal ini$a$on. ü Differences in gene expression between prokaryotes
and eukaryotes. •  Rela%onship of DNA muta%on to protein structure and func%on. Sec$on 8 Learning Goals •  Explain the molecular mechanism by which transcrip$onal and transla$onal inhibitors kill bacteria. •  Indicate the key molecules that interact with the ribosome and the func$on of each interac$on. •  Provide a molecular explana$on for how the transcrip$onal and transla$onal inhibitors achieve prokaryo$c specificity. •  Compare a silent, nonsense and missense muta$on in terms of DNA altera$on, result on mRNA structure and polypep$de structure and func$on. •  Explain how point muta$ons in a gene might result in a func$onal, yet structurally different gene product. Tetracycline inhibits transla$on •  We know that an$bio$cs act through their specific, non-­‐covalent binding interac$ons with the target molecule. •  Tetracycline binds to its target to inhibit transla$on. •  Consider what molecules tetracycline might bind to exert its effect. Consider what interac$ons might be blocked by tetracycline interac$ng with its target hUp://upload.wikimedia.org/wikipedia/commons/b/b1/Ribosome_mRNA_transla$on_en.svg •  Tetracycline binds to the 30S (small) subunit nucleo$des through non-­‐covalent bonding interac$ons •  The binding alters the structure of the small subunit such that the tRNA an$codon and mRNA codon interac$ons are obscured hUp://faculty.ccbcmd.edu/courses/bio141/
lecguide/unit2/control/tetres.html Isn’t transla$on a highly conserved process? – Is the target molecule present in both prokaryotes and eukaryotes? •  If so, how does tetracycline achieve prokaryo$c specificity? •  If not, how does the mechanism of transla$on differ in prokaryotes vs eukaryotes? To understand tetracycline binding specificity and implica$ons, addi$onal knowledge of ribosome structure is necessary The ribosome consists of rRNA and a few small polypep$des •  Just as polypep$des use non-­‐covalent bonding interac$ons to adopt func$onal folded structure (conforma$on), RNA molecules can also exert intra-­‐
molecular non-­‐covalent bonding, primarily in the form of hydrogen bonds between complementary base pairs. •  The rRNA associates with the small polypep$des through non-­‐covalent bonding interac$ons. •  This “folded” structure of the rRNA + polypep$des confers the shape and func$on of the molecule. •  It is the RNA regions of the ribosome, not polypep$des, that confer cataly$c proper$es. This figure shows the general shape of the whole ribosome If we zoom in on the RNA por$on, we see that individual nucleo$des undergo base-­‐pairing to form “stems” and “loops” hUp://rna.ucsc.edu/rnacenter/images/figs/ecoli_23s.jpg The molecule, complete with base-­‐pairing interac$ons, undergoes further folding resul$ng in the ter$ary, ac$ve structure of the ribosome hUp://rna.ucsc.edu/rnacenter/images/figs/70s_atrna_labels.jpg •  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$cs, such as tetracycline, bind to the 30S or 50S ribosomal subunit. Where on these subunits do you think they bind? one edge and either hydrophobic or stacking interactions with the other side. We have found two binding
sites for Tc within the small ribosomal subunit. The better occupied site is located near the acceptor site for
aminoacylated tRNA between the head and the body of
the 30S (the A site), and the less occupied site is at the
interface between three RNA domains in the body of
the subunit. Throughout this paper, the binding site of
tetracycline near the A site is termed the primary site,
whereas the location in the body is called the secondary
binding site. The discovery of a second binding site is
not surprising since the drug is known to have secondary, possibly nonspecific sites on both subunits (Pestka,
1974; Vazquez, 1974).
The an$bio$cs shown here bind to the 16S rRNA of the 30S (small) subunit Figure 1. Overview of Tetracycline, Pactamycin, and Hygromycin B in the 30S Subunit
Pactamycin The parts of the 16S RNA that make contact with any of the an$bio$cs are colored Antibiotics are shown as space-filling models, with tetracycline (blue), pactamycin
(green), and hygromycin B (red). The parts of
16S RNA that make contacts with any of the
three antibiotics are colored as in the following figures.
Hygromycin B Tetracycline 30S (small) subunit From Broderson (2000) Cell 103:1143-­‐ 1154 The primary binding site of tetracycline (yellow) is to the A site of the ribosome Green, blue and cyan represent relevant helices of the 16S rRNA tetracycline From Broderson (2000) Cell 103:1143-­‐ 1154 hUp://commons.wikimedia.org/wiki/
File:Tetracycline_structure.svg tetracycline Tetracycline binding likely interferes with tRNA (red) and mRNA (yellow) access and codon-­‐
an$codon interac$on From Broderson (2000) Cell 103:1143-­‐ 1154 6
Model depic$ng possible tetracycline binding interac$ons with 16S rRNA The point here is not to memorize this specific example, but to use it as context to understand: •  The role of non-­‐covalent bonding interac$ons in biology •  The rela$onship of structure to func$on •  How the ribosome func$ons at the molecular level tetracycline Broderson (2000) Cell 103:1143-­‐ 1154 Sec$on 8 Learning Goals •  Explain the molecular mechanism by which transcrip$onal and transla$onal inhibitors kill bacteria. ü Indicate the key molecules that interact with the ribosome and the func$on of each interac$on. •  Explain how the transcrip$onal and transla$onal inhibitors achieve prokaryo$c specificity. •  Compare a silent, nonsense and missense muta$on in terms of DNA altera$on, result on mRNA structure and polypep$de structure and func$on. •  Explain how point muta$ons in a gene might result in a func$onal, yet structurally different gene product. Revisi$ng our earlier ques$on… – Is the target molecule present in both prokaryotes and eukaryotes? •  If so, how does tetracycline achieve prokaryo$c specificity? •  If not, how does the mechanism of transla$on differ in prokaryotes vs eukaryotes? •  Cell wall synthesis inhibitors and sulfonamides achieve specificity by targe$ng enzymes not present in mammalian cells. •  Gramicidin affects mammalian cell membranes so is only used topically. •  How does tetracycline achieve prokaryo$c specificity? Interpret this graph [3H]Tet refers to tetracycline in which hydrogen is replaced with the radioac$ve [3H] form. Budkevich T V et al. Nucl. Acids Res. 2008;36:4736-4744
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. Budkevich T V et al. Nucl. Acids Res. 2008;36:4736-4744
•  Tetracycline does bind to eukaryo$c ribosomes, but with lower affinity than it binds to prokaryo$c ribosomes. •  What, at the molecular level, might account for this difference in binding affinity? 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$cs Lafontaine and Tollervey (2001)Nature Reviews Molecular Cell Biology 2, 514-­‐520 V1 V2 V3 V4 V5 V6 tetracycline Broderson (2000) Cell 103:1143-­‐ 1154 V7 1294 1243 1173 6
1117 986 Numbers refer to single nucleo$de posi$ons in the 16S rRNA 1043 Tetracycline binding 1054 1197-­‐1198 V8 V9 V10
•  The small and large ribosomal subunit RNA each have both highly conserved and variable regions in their nucleo$de sequence. •  The proposed binding site for tetracycline is in a conserved region. •  Therefore, binding affinity differences must be aUributable to single nucleo$de differences between prokaryote and eukaryote 16S/18S rRNA amidst the otherwise highly conserved sequence. •  These are very special nucleo%de changes that retain func%on. Transcrip$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 hUp://www.cpbs.cnrs.fr/spip.php?ar$cle81&lang=fr •  The protein synthesis inhibitors achieve specificity due to subtle structural differences in binding site between prokaryo$c and eukaryo$c target. This is also true for the transcrip$on inhibitors such as rifampicin. •  The subtle structural differences result from differences in DNA sequence of the coding genes. •  Even subtle differences in structure can result in altered binding affinity of an$bio$c for target. Sec$on 8 Learning Goals •  Explain the molecular mechanism by which transcrip$onal and transla$onal inhibitors kill bacteria. ü  Indicate the key molecules that interact with the ribosome and the func$on of each interac$on. ü  Provide a molecular explana$on for how the transcrip$onal and transla$onal inhibitors achieve prokaryo$c specificity. •  Compare a silent, nonsense and missense muta$on in terms of DNA altera$on, result on mRNA structure and polypep$de structure and func$on. •  Explain how point muta$ons in a gene might result in a func$onal, yet structurally different gene product. How do these nucleo$de changes arise and why are they maintained? DNA polymerase makes an occasional mistake 5’ GTGAAT
3’ CACTTA
DNA polymerase makes an occasional mistake And when it does, repair enzymes are likely to catch it and repair it Those that go unno$ced become permanent muta$ons auer the next cell division 5’ GTGAAT
3’ CACTTA
We use the logic of complementary base-­‐pairing and the gene$c code to predict polypep$de sequence: 5’ ATG GCT TGC 3’
3’ TAC CGA ACG 5’
DNA transcrip$on 5’ AUGGCUUGC 3’
mRNA transla$on N Met–Ala–Cys C
polypep$de What would be the effect on the polypep$de if there was a single nucleo$de change in the DNA sequence: 5’ ATG GCT TGC 3’
DNA 3’ TAC CGA ACG 5’
Muta$onal change 5’ ATG TCT TGC 3’
3’ TAC CGA ACG 5’
Cell division 5’ ATG GCT TGC 3’ + 5’ ATG TCT TGC 3’ DNA 3’ TAC AGA ACG 5’
3’ TAC CGA ACG 5’
A serine residue is subs$tuted for alanine 5’ ATG GCT TGC 3’
DNA 3’ TAC CGA ACG 5’
transcrip$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$on N Met–Ser–Cys C
•  Are the proper$es of alanine and serine similar? •  Do you think that this single amino acid subs$tu$on will affect the conforma$on of the polypep$de? •  If so, how? If not, why? •  If conforma$on is altered, will func$on be altered? Alanine WikiCommons user: Borb Serine There are different classes of DNA sequence muta$ons •  Point muta$ons: single nucleo$de subs$tu$on •  Dele$ons: one or more nucleo$des lost •  Inser$ons: one or more nucleo$des gained We can use a three-letter word analogy to consider the
effects of some DNA mutations on amino acid sequence
HIT THE BAT
Normal “polypeptide” sequence
HIT CTH EBA T
Single nucleotide insertion
RESULT: “frameshift” of codon usage
HIT CAT THE BAT
Three nucleotide insertion
RESULT: single amino acid inserted; codon
usage remains “in frame”
HIT CHE BAT
Single “nucletide” substitution (point
mutation)
RESULT: silent, missense or nonsense
(see next slide)
Classes of point muta$ons NORMAL SILENT NONSENSE MISSENSE TGC
ACG
TGT
ACA
TGA
ACT
TGG
ACC
transcrip$on UGC
transla$on Cys
Correct amino acid transcrip$on UGU
transla$on Cys
No change in amino acid sequence transcrip$on UGA
transla$on STOP
Premature STOP codon introduced transcrip$on UGG
transla$on Trp
Single amino acid change Point muta$ons drive evolu$on •  May destroy func$on of product/protein (“null”) •  May slightly alter structureà slightly alter func$on - Par$al loss of func$on - Gain of func$on The following slide series has been modified from the original that was developed as part of a “teachable $dbit” at the 2012 Northeast Regional Summer Ins$tute More informa$on and the slide set in its en$rety can be found at the Yale Center for Scien$fic Teaching website: hUp://cst.yale.edu/biology-­‐and-­‐chemistry-­‐interface-­‐$dbits Authors: Carol Bascom-­‐Slack, Carlton Cooper, Michal Hallside, Jaqui Johnson, Gillian Phillips, Eugenia Ribeiro-­‐Hurley, and Erica Selva. Examina$on of Red Blood Cells Sickle Cell Anemia Normal 2012 Northeast Regional Summer Ins$tute Hemoglobin • 
• 
• 
• 
A principal protein component of red blood cells Tetramer composed of two α-­‐subunits and two β-­‐subunits α-­‐subunits have 141 amino acids while the β-­‐subunits have 146 amino acids Each subunit carries oxygen 2012 Northeast Regional Summer Ins$tute Think-­‐Pair-­‐Share 1. Which amino acid side chains (R groups) are hydrophobic? 2. Which amino acid side chains (R groups) are hydrophilic? Valine (Val, V) Glutamate (Glu, E) Serine (Ser, S) Leucine (Leu, L) Lysine (Lys, K) Threonine (Thr, T) 2012 Northeast Regional Summer Ins$tute Think-­‐Pair-­‐Share Valine (Val, V) !
Glutamate (Glu, E) !
Serine (Ser, S)!
Leucine (Leu, L)!
Lysine (Lys, K) !
Threonine (Thr, T)!
Hydrophobic amino acid R-­‐group (side chain) Hydrophilic amino acid R-­‐group (side chain) 2012 Northeast Regional Summer Ins$tute Think pair share: Valine (Val, V) Glutamate (Glu, E) Serine (Ser, S) Leucine (Leu, L) Lysine (Lys, K) Threonine (Thr, T) Hydrophobic amino acid R-­‐group (side chain) Hydrophilic amino acid R-­‐group (side chain) 2012 Northeast Regional Summer Ins$tute 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 Ins$tute Each blue dot=one amino acid α=141 amino acids β=146 amino acids β α α β 2012 Northeast Regional Summer Ins$tute 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 2012 Northeast Regional Summer Ins$tute Where in this folded protein might this change have occurred? A. Near the oxygen binding site B. At the interface between subunits C. On the surface •  In this case, the correct answer is (B) •  But all would be logical predic$ons •  The answer can only be determined through experimental analyses 2012 Northeast Regional Summer Ins$tute These are the loca$ons of the changed amino acids. Glutamate (hydrophilic) Valine (hydrophobic ) 2012 Northeast Regional Summer Ins$tute The single amino acid subs$tu$on causes hemoglobin to aggregate resul$ng in the sickle shaped red blood cells 2012 Northeast Regional Summer Ins$tute Remember: An external hydrophilic amino acid is replaced with a hydrophobic one Glutamate (hydrophilic) Valine (hydrophobic) 2012 Northeast Regional Summer Ins$tute Sec$on 8 Learning Goals ü  Explain the molecular mechanism by which transcrip$onal and transla$onal inhibitors kill bacteria. ü  Indicate the key molecules that interact with the ribosome and the func$on of each interac$on. ü  Provide a molecular explana$on for how the transcrip$onal and transla$onal inhibitors achieve prokaryo$c specificity. ü  Compare a silent, nonsense and missense muta$on in terms of DNA altera$on, result on mRNA structure and polypep$de structure and func$on. ü  Explain how point muta$ons in a gene might result in a func$onal, yet structurally different gene product. Transcrip$on and Transla$on Review Think-­‐Pair-­‐Share •  Consider the effects of a nucleo$de synthesis error that results in complete loss of protein func$on and that the protein is required for life of the cell. –  First, consider the effect on an individual prokaryo$c cell if the muta$on was introduced by DNA polymerase in DNA replica$on. –  Next, consider the effect on an individual prokaryo$c cell if the muta$on was introduced by RNA polymerase in transcrip$on. •  Consider the effects of a nucleo$de synthesis error that results in complete loss of protein func$on and that the protein is required for life of the cell. –  First, consider the effect on an individual prokaryo$c cell if the muta$on was introduced by RNA polymerase in transcrip$on. –  Next, consider the effect on an individual prokaryo$c cell if the muta$on was introduced by DNA polymerase in DNA replica$on. Would your answer change if the errors took place in a eukaryote? Compare the effects of an error in transcrip$on. . . Modified from Figure 7-2 Essential Cell Biology (© Garland Science 2010)
With an error in DNA replica$on Modified from Figure 7-2 Essential Cell Biology (© Garland Science 2010)
RNA polymerase and RNA synthesis doesn’t require the same high level of fidelity as DNA polymerase and DNA synthesis •  Consider the effects of a nucleo$de synthesis error that results in complete loss of protein func$on and that the protein is required for life of the cell. –  First, consider the effect on an individual prokaryo$c cell if the muta$on was introduced by RNA polymerase in transcrip$on. –  Next, consider the effect on an individual prokaryo$c cell if the muta$on was introduced by DNA polymerase in DNA replica$on. Would your answer change if the errors took place in a eukaryote? •  Muta$ons in the soma$c cells are not heritable, but lead to disease. •  Mammals have two copies of each chromosome (diploid) and only one is acquired by each gamete. Therefore, a par$cular muta$on may not be inherited. •  Every muta$on in bacteria is heritable Summary Statements •  An$bio$cs must target essen$al (conserved) func$ons, yet they must be prokaryote-­‐specific to be most useful. •  Specificity is achieved in two main ways: –  An$bio$c targets a molecule that is essen$al for prokaryotes but not for eukaryotes. –  An$bio$c targets a structural change in a conserved molecule. The structural difference is the result of nucleo$de differences between prokaryote and eukaryote DNA that alters structure of the gene product but retains func$on.