Sec$on 8. Gene$c Muta$ons, Ribosome Structure and the Tetracyclines

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1 Sec$on 8 Gene$c Muta$ons, Ribosome Structure and the Tetracyclines

2 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.

3 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.

4 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.

5 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.

6 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

7 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

8 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?

9 To understand tetracycline binding specificity and implica$ons, addi$onal knowledge of ribosome structure is necessary

10 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.

11 This figure shows the general shape of the whole ribosome

12 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

13 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

14 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?

15 The an$bio$cs shown here bind to the 16S rrna of the 30S (small) subunit The parts of the 16S RNA that make contact with any of the an$bio$cs are colored Pactamycin Tetracycline Hygromycin B 30S (small) subunit From Broderson (2000) Cell 103:

the 16S rrna tetracycline From Broderson (2000) Cell 103:1143-1154

16 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: hup://commons.wikimedia.org/wiki/ File:Tetracycline_structure.svg

17 tetracycline Tetracycline binding likely interferes with trna (red) and mrna (yellow) access and codon- an$codon interac$on From Broderson (2000) Cell 103:

18 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:

19 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.

20 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?

21 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?

22 Interpret this graph [ 3 H]Tet refers to tetracycline in which hydrogen is replaced with the radioac$ve [ 3 H] form. Budkevich T V et al. Nucl. Acids Res. 2008;36:

23 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:

24 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?

25 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,

V1 V2 V3 V4 V5 V6 tetracycline Broderson (2000) Cell 103:1143-1154 V7 1294 1243 1173 6 1117 986

26 V1 V2 V3 V4 V5 V6 tetracycline Broderson (2000) Cell 103: V Numbers refer to single nucleo$de posi$ons in the 16S rrna 1043 Tetracycline binding V8 V9 V10

27 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.

28 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://

29 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.

30 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.

31 How do these nucleo$de changes arise and why are they maintained?

32 DNA polymerase makes an occasional mistake 5 GTGAAT 3 CACTTA

repair it Those that go unno$ced become permanent

33 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

34 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

35 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 3 TAC CGA ACG 5 DNA Muta$onal change 5 ATG TCT TGC 3 3 TAC CGA ACG 5 Cell division 5 ATG GCT TGC 3 3 TAC CGA ACG ATG TCT TGC 3 3 TAC AGA ACG 5 DNA

36 A serine residue is subs$tuted for alanine DNA 5 ATG GCT TGC 3 3 TAC CGA ACG 5 5 ATG TCT TGC 3 3 TAC AGA ACG 5 transcrip$on 5 AUGGCUUGC 3 transcrip$on 5 AUGUCUUGC 3 transla$on transla$on N Met Ala Cys C N Met Ser Cys C

37 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 Serine WikiCommons user: Borb

38 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

39 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 HIT CAT THE BAT HIT CHE BAT Single nucleotide insertion RESULT: frameshift of codon usage Three nucleotide insertion RESULT: single amino acid inserted; codon usage remains in frame Single nucletide substitution (point mutation) RESULT: silent, missense or nonsense (see next slide)

40 Classes of point muta$ons NORMAL SILENT NONSENSE MISSENSE TGC ACG transcrip$on TGT ACA transcrip$on TGA ACT transcrip$on TGG ACC transcrip$on UGC UGU UGA UGG transla$on transla$on transla$on transla$on Cys Cys STOP Trp Correct amino acid No change in amino acid sequence Premature STOP codon introduced Single amino acid change

41 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

42 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.

43 2012 Northeast Regional Summer Ins$tute Examina$on of Red Blood Cells Sickle Cell Anemia Normal

44 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

45 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)

46 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

Hydrophobic amino acid R- group (side chain) Hydrophilic amino

47 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

48 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

49 Each blue dot=one amino acid α=141 amino acids β=146 amino acids β α α β 2012 Northeast Regional Summer Ins$tute

50 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

51 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

52 2012 Northeast Regional Summer Ins$tute These are the loca$ons of the changed amino acids. Glutamate (hydrophilic) Valine (hydrophobic )

53 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

54 Remember: An external hydrophilic amino acid is replaced with a hydrophobic one 2012 Northeast Regional Summer Ins$tute Glutamate (hydrophilic) Valine (hydrophobic)

55 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.

56 Transcrip$on and Transla$on Review

57 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.

58 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?

59 Compare the effects of an error in transcrip$on... Modified from Figure 7-2 Essential Cell Biology ( Garland Science 2010)

60 With an error in DNA replica$on Modified from Figure 7-2 Essential Cell Biology ( Garland Science 2010)

61 RNA polymerase and RNA synthesis doesn t require the same high level of fidelity as DNA polymerase and DNA synthesis

62 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?

63 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

64 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.

Section 8: Genetic Mutations, Ribosome Structure,

Section 8: Genetic Mutations, Ribosome Structure, and Tetracycline TOPICS Ribosome structure Classes of mutation Mechanism of action of the tetracyclines SUMMARY Up to this point, our focus has been on