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Animation: Transcription Introduction

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1 Animation: Transcription Introduction
© 2016 Pearson Education, Inc.

2 © 2016 Pearson Education, Inc.
Promoter Transcription unit 5¢ 3¢ 3¢ 5¢ DNA Start point RNA polymerase Initiation 5¢ 3¢ 3¢ 5¢ RNA transcript Template strand of DNA Unwound DNA Elongation Rewound DNA 5¢ 3¢ 3¢ 3¢ 5¢ 5¢ Figure 14.8-s3 The stages of transcription: initiation, elongation, and termination (step 3) RNA transcript Direction of transcription (“downstream”) Termination 5¢ 3¢ 3¢ 5¢ 5¢ 3¢ Completed RNA transcript © 2016 Pearson Education, Inc.

3 © 2016 Pearson Education, Inc.
Promoter Nontemplate strand DNA 5¢ T A T A A A A 3¢ 3¢ A T A T T T T 5¢ A eukaryotic promoter TATA box Start point Template strand Transcription factors 5¢ 3¢ Several transcription factors bind to DNA. 3¢ 5¢ RNA polymerase II Transcription factors Figure 14.9 The initiation of transcription at a eukaryotic promoter Transcription initiation complex forms. 5¢ 3¢ 3¢ 3¢ 5¢ 5¢ RNA transcript Transcription initiation complex © 2016 Pearson Education, Inc.

4 protein-coding segments
A modified guanine nucleotide added to the 5¢ end 50–250 adenine nucleotides added to the 3¢ end Region that includes protein-coding segments Polyadenylation signal 5¢ 3¢ G … P P P AAUAAA AAA AAA Start codon Stop codon 5¢ Cap Figure RNA processing: addition of the 5 cap and poly-A tail 5¢ UTR 3¢ UTR Poly-A tail © 2016 Pearson Education, Inc.

5 © 2016 Pearson Education, Inc.
Amino acid attachment site 5¢ 3¢ Hydrogen bonds Figure The structure of transfer RNA (tRNA) (part 2: three-dimensional structure) A A G 3¢ 5¢ Anticodon Anticodon (b) Three-dimensional structure (c) Symbol used in this book © 2016 Pearson Education, Inc.

6 © 2016 Pearson Education, Inc.
Amino acid and tRNA enter active site. Tyrosine (Tyr) (amino acid) Tyrosyl-tRNA synthetase Tyr-tRNA A U A ATP Aminoacyl-tRNA synthetase Complementary tRNA anticodon AMP + 2 P i tRNA Figure Aminoacyl-tRNA synthetases provide specificity in joining amino acids to their tRNAs. Using ATP, synthetase catalyzes covalent bonding. Amino acid Aminoacyl tRNA released. Computer model © 2016 Pearson Education, Inc.

7 © 2016 Pearson Education, Inc.
Amino end of polypeptide Codon recognition E 3¢ mRNA Ribosome ready for next aminoacyl tRNA P site A site 5¢ GTP GDP + P i E E P A P A Figure s3 The elongation cycle of translation (step 3) GDP + P i Translocation Peptide bond formation GTP E P A © 2016 Pearson Education, Inc.

8 Protein Folding and Post-Translational Modifications
Remember protein structure? Secondary, tertiary, quaternary? Some proteins need prosthetic groups attached Heme group on hemoglobin © 2016 Pearson Education, Inc. 8 8

9 Targeting Polypeptides to Specific Locations
Eukaryotic cells have free and bound ribosomes Free synthesizes proteins that function in the cytosol Bound ribosomes Secreted proteins Membrane proteins Lysosomal proteins © 2016 Pearson Education, Inc. 9 9

10 Polypeptide synthesis always begins in the cytosol
finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER or for secretion are marked by a signal peptide A signal-recognition particle (SRP) binds to the signal peptide The SRP brings the signal peptide and its ribosome to the ER © 2016 Pearson Education, Inc. 10 10

11 © 2016 Pearson Education, Inc.
Polypeptide synthesis begins. SRP binds to signal peptide. SRP binds to receptor protein. SRP detaches and polypeptide synthesis resumes. Signal- cleaving enzyme cuts off signal peptide. Completed polypeptide folds into final conformation. Ribosome mRNA Signal peptide ER membrane Signal peptide removed SRP Figure The signal mechanism for targeting proteins to the ER Protein CYTOSOL SRP receptor protein ER LUMEN Translocation complex © 2016 Pearson Education, Inc.

12 © 2016 Pearson Education, Inc.
Completed polypeptide Growing polypeptides Incoming ribosomal subunits Polyriboso me Start of mRNA (5¢ end) End of mRNA (3¢ end) (a) An mRNA molecule translated simultaneously by several ribosomes Figure Polyribosomes Ribosomes mRNA (b) A large polyribosome in a bacterial cell (TEM) 0.1 m © 2016 Pearson Education, Inc.

13 © 2016 Pearson Education, Inc.
Figure 14.23 RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 m RNA polymerase DNA Figure Coupled transcription and translation in bacteria Polyribosome Polypeptide (amino end) Ribosome mRNA (5¢ end) © 2016 Pearson Education, Inc.

14 © 2016 Pearson Education, Inc.
Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 3¢ Figure The anatomy of a functioning ribosome (part 3: mRNA and tRNA) Codons 5¢ (c) Schematic model with mRNA and tRNA © Pearson Education, Inc. © 2016 Pearson Education, Inc.

15 What Is a Gene? A gene can be defined as a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule © 2016 Pearson Education, Inc. 15 15

16 © 2016 Pearson Education, Inc.
Mutations What are they? Point mutations are chemical changes in just one nucleotide pair of a gene What if it occurs in a somatic cell? What if it occurs in a gamete? Germ-line mutations © 2016 Pearson Education, Inc. 16 16

17 Does it change the protein?
Wild-type b-globin Sickle-cell b-globin Wild-type b-globin DNA Mutant b-globin DNA 3¢ C T C 5¢ 3¢ C A C 5¢ 5¢ G A G 3¢ 5¢ G T G 3¢ mRNA mRNA 5¢ G A G 3¢ 5¢ G U G 3¢ Figure The molecular basis of sickle-cell disease: a point mutation Normal hemoglobin Sickle-cell hemoglobin Glu Val © 2016 Pearson Education, Inc.

18 Types of Small-Scale Mutations
Point mutations within a gene can be divided into two general categories Single nucleotide-pair substitutions Nucleotide-pair insertions or deletions © 2016 Pearson Education, Inc. 18 18

19 © 2016 Pearson Education, Inc.
Substitutions replaces one nucleotide and its partner with another pair of nucleotides Silent mutations no effect on the amino acid produced by a codon because of redundancy in the genetic code © 2016 Pearson Education, Inc. 19 19

20 © 2016 Pearson Education, Inc.
Wild type DNA template strand 3¢ T A C T T C A A A C C G A T T 5¢ 5¢ A T G A A G T T T G G C T A A 3¢ mRNA 5¢ A U G A A G U U U G G C U A A 3¢ Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair substitution: silent A instead of G 3¢ T A C T T C A A A C C A A T T 5¢ Figure Types of small-scale mutations that affect mRNA sequence (part 1: silent) 5¢ A T G A A G T T T G G T T A A 3¢ U instead of C 5¢ A U G A A G U U U G G U U A A 3¢ Met Lys Phe Gly Stop © 2016 Pearson Education, Inc.

21 © 2016 Pearson Education, Inc.
Wild type DNA template strand 3¢ T A C T T C A A A C C G A T T 5¢ 5¢ A T G A A G T T T G G C T A A 3¢ mRNA 5¢ A U G A A G U U U G G C U A A 3¢ Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair substitution: missense T instead of C 3¢ T A C T T C A A A T C G A T T 5¢ Figure Types of small-scale mutations that affect mRNA sequence (part 2: missense) 5¢ A T G A A G T T T A G C T A A 3¢ A instead of G 5¢ A U G A A G U U U A G C U A A 3¢ Met Lys Phe Ser Stop © 2016 Pearson Education, Inc.

22 Missense mutations = wrong amino acid
Substitution mutations are usually missense mutations © 2016 Pearson Education, Inc. 22 22

23 © 2016 Pearson Education, Inc.
Wild type DNA template strand 3¢ T A C T T C A A A C C G A T T 5¢ 5¢ A T G A A G T T T G G C T A A 3¢ mRNA 5¢ A U G A A G U U U G G C U A A 3¢ Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair substitution: nonsense A instead of T 3¢ T A C A T C A A A C C G A T T 5¢ Figure Types of small-scale mutations that affect mRNA sequence (part 3: nonsense) 5¢ A T G T A G T T T G G C T A A 3¢ U instead of A 5¢ A U G U A G U U U G G U U A A 3¢ Met Stop © 2016 Pearson Education, Inc.

24 Nonsense mutations = premature stop codon
nearly always leading to a nonfunctional protein © 2016 Pearson Education, Inc. 24 24

25 © 2016 Pearson Education, Inc.
Wild type DNA template strand 3¢ T A C T T C A A A C C G A T T 5¢ 5¢ A T G A A G T T T G G C T A A 3¢ mRNA 5¢ A U G A A G U U U G G C U A A 3¢ Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair insertion: frameshift causing immediate nonsense Extra A 3¢ T A C A T T C A A A C C G A T T 5¢ Figure Types of small-scale mutations that affect mRNA sequence (part 4: frameshift, nonsense) 5¢ A T G T A A G T T T G G C T A A 3¢ Extra U 5¢ A U G U A A G U U U G G C U A A 3¢ Met Stop © 2016 Pearson Education, Inc.

26 © 2016 Pearson Education, Inc.
Wild type DNA template strand 3¢ T A C T T C A A A C C G A T T 5¢ 5¢ A T G A A G T T T G G C T A A 3¢ mRNA 5¢ A U G A A G U U U G G C U A A 3¢ Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair deletion: frameshift causing extensive missense missing A 3¢ T A C T T C A A C C G A T T 5¢ Figure Types of small-scale mutations that affect mRNA sequence (part 5: frameshift, missense) 5¢ A T G A A G T T G G C T A A 3¢ U missing 5¢ A U G A A G U U G G C U A A 3¢ Met Lys Leu Ala © 2016 Pearson Education, Inc.

27 © 2016 Pearson Education, Inc.
Wild type DNA template strand 3¢ T A C T T C A A A C C G A T T 5¢ 5¢ A T G A A G T T T G G C T A A 3¢ mRNA 5¢ A U G A A G U U U G G C U A A 3¢ Protein Met Lys Phe Gly Stop Amino end Carboxyl end 3 nucleotide-pair deletion: no frameshift, but one amino acid missing T T C missing 3¢ T A C A A A C C G A T T 5¢ Figure Types of small-scale mutations that affect mRNA sequence (part 6: missing amino acid) 5¢ A T G T T T G G C T A A 3¢ A A G missing 5¢ A U G U U U G G C U A A 3¢ Met Phe Gly Stop © 2016 Pearson Education, Inc.

28 Insertions and Deletions
Insertions and deletions are additions or losses of nucleotide pairs in a gene Much more detrimental than substitutions Why?????? may alter the reading frame of the genetic message frameshift mutation © 2016 Pearson Education, Inc. 28 28

29 New Mutations and Mutagens
Spontaneous mutations can occur during DNA replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations Most cancer-causing chemicals (carcinogens) are mutagenic, and the converse is also true © 2016 Pearson Education, Inc. 29 29

30 Alterations of chromosome number or structure cause some genetic disorders
often lead to spontaneous abortions (miscarriages) or cause a variety of developmental disorders Plants tolerate such genetic changes better than animals do © 2016 Pearson Education, Inc. 30

31 Abnormal Chromosome Number
In nondisjunction, pairs of homologous chromosomes do not separate normally during meiosis one gamete receives two of the same type of chromosome, and another gamete receives no copy If this gamete is involved in fertilization Aneuploid individual results Ex: Down Syndrom © 2016 Pearson Education, Inc. 31

32 Video: Nondisjunction
© 2016 Pearson Education, Inc.

33 Nondisjunction of homologous chromosomes in meiosis I
Figure s3 Meiosis I Nondisjunction Meiosis II Non- disjunction Gametes Figure s3 Meiotic nondisjunction (step 3) n  1 n  1 n  1 n  1 n  1 n  1 n n Number of chromosomes Nondisjunction of homologous chromosomes in meiosis I (b) Nondisjunction of sister chromatids in meiosis II © 2016 Pearson Education, Inc.

34 A monosomic zygote has only one copy of a particular chromosome
A trisomic zygote has three copies of a particular chromosome © 2016 Pearson Education, Inc. 34

35 Alterations of Chromosome Structure
Breakage of a chromosome can lead to four types of changes in chromosome structure Deletion removes a chromosomal segment Duplication repeats a segment Inversion reverses orientation of a segment within a chromosome Translocation moves a segment from one chromosome to another © 2016 Pearson Education, Inc. 35

36 (a) Deletion A B C D E F G H A deletion removes a chromosomal segment.
Figure (a) Deletion A B C D E F G H A deletion removes a chromosomal segment. A B C E F G H (b) Duplication Figure Alterations of chromosome structure (part 1: deletion and duplication) A B C D E F G H A duplication repeats a segment. A B C B C D E F G H © 2016 Pearson Education, Inc.

37 An inversion reverses a segment within a chromosome.
Figure (c) Inversion A B C D E F G H An inversion reverses a segment within a chromosome. A D C B E F G H (d) Translocation A B C D E F G H M N O P Q R Figure Alterations of chromosome structure (part 2: inversion and translocation) A translocation moves a segment from one chromosome to a nonhomologous chromosome. M N O C D E F G H A B P Q R © 2016 Pearson Education, Inc.

38 Duplications and translocations also tend to be harmful
An embryo with a large deletion is likely missing a number of essential genes generally lethal Duplications and translocations also tend to be harmful In inversions, the balance of genes is normal but phenotype may be influenced if the expression of genes is altered © 2016 Pearson Education, Inc. 38

39 Figure 12.15 Figure Down syndrome © 2016 Pearson Education, Inc.

40 Disorders Caused by Structurally Altered Chromosomes
Certain cancers, including chronic myelogenous leukemia (CML), are caused by translocations of chromosomes © 2016 Pearson Education, Inc. 40

41 Translocated chromosome 22 (Philadelphia chromosome)
Figure 12.16 Normal chromosome 9 Normal chromosome 22 Reciprocal translocation Figure Translocation associated with chronic myelogenous leukemia (CML) Translocated chromosome 9 Translocated chromosome 22 (Philadelphia chromosome) © 2016 Pearson Education, Inc.

42 Analyzing DNA using RFLP analysis
Based on use of restriction endonucleases

43

44

45

46

47 BglII (lane 1) and BstEII (Lane 2) each cut pTut once
BglII (lane 1) and BstEII (Lane 2) each cut pTut once. The result in each case is a single frament of 7666 bp. Therefore we can draw very simple restriction maps for these two enzymes: (Note: This does not mean the sites are in the same place, as we will see.)

48 Use DNA fingerprinting data to make a restriction map
Circular plasmid is 7,666 Base pairs (bp) in length

49 BglII and BstEII together produce two fragments of 6008 and 1658 bp
BglII and BstEII together produce two fragments of 6008 and 1658 bp. Thus the two sites can be placed on a map as follows: Conceptually, you can visualize how we made this map by considering two disks, one with the BglII site, and the other with the BstEII site that we overlaid and rotated until BglII and BstEII were 1658 bp apart. [The map, of course, could be drawn as a mirror image.]

50 EcoRV cuts pTut two times to produce fragments of 4729 and 2937 bp
EcoRV cuts pTut two times to produce fragments of 4729 and 2937 bp. The EcoRV map, again without putting the sites at absolute locations.

51 EcoRV and BglII together produce three fragments of 4729, 1992, and 945 bp. Comparing this with EcoRV cutting alone, we can conclude that the BglII site must be in the 2937 bp EcoRV fragment, cutting it into the 1992 and 945 bp pieces. Using our rotating disks method, we can create an EcoRV and BglII map.

52 EcoRV and BstEII together produce three fragments of 4016, 2937, and 713 bp. Comparing this with EcoRV cutting alone, we can conclude that the BstEII site must be in the 4729 bp EcoRV fragment, cutting it into the 4016 and 713 bp fragments. Using our rotating disk method, we can create an EcoRV and BstEII map

53 If we look again at the BglII + BstEII cut data, we see that the fragments produced are 6008 and 1658 bp. So, we need to overlay the EcoRV + BglII and EcoRV + BstEII maps to see how this can be done. If you try this, you will see it will not work as the maps are drawn. The next page shows how an alternative mirror image map for EcoRV + BstEII can help us.

54 If we overlay the EcoRV + BglII map with this new EcoRV + BstEII map, we can make the two mesh to be compatible with the digest data

55 © 2016 Pearson Education, Inc.
Figure 14.UN02 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.UN02 In-text figure, transcription, p. 285 TRANSLATION Ribosome Polypeptide © 2016 Pearson Education, Inc.

56 © 2016 Pearson Education, Inc.
Figure 14.UN03 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.UN03 In-text figure, RNA processing, p. 287 TRANSLATION Ribosome Polypeptide © Pearson Education, Inc. © 2016 Pearson Education, Inc.

57 © 2016 Pearson Education, Inc.
Figure 14.UN04 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Figure 14.UN04 In-text figure, translation, p. 289 Polypeptide © Pearson Education, Inc. © 2016 Pearson Education, Inc.

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