Topic 2.7 Replication, Transcription & Translation

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1 Topic 2.7 Replication, Transcription & Translation

2 INTRO The central dogma of biology describes how information from DNA is able to influence the traits of an organism. There are three processes involved: 2

3 2.7 A DNA Replication

4 INTRO In order for cells to reproduce and pass on genes, they must copy 4 their genome in a process called DNA replication. This occurs during the S phase of the cell cycle.

5 Understandings U2: Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds. In order for DNA to be replicated, the hydrogen bonds between the two strands must be broken. This is done by an enzyme called helicase, which exposes the nitrogenous bases. 5 Helicase

6 Understandings U3: DNA polymerase links nucleotides together to form a new strand, using the preexisting strand as a template. After the strands are separated, DNA polymerase links together nucleotides complementary (A-T & C-G) to the original strands. This results in two new DNA molecules identical to the original. 6 DNA polymerase

7 7 Understandings U1: The replication of DNA is semiconservative and depends on complementary base pairing. Because of complementary base pairing, the daughter DNA molecules are identical to the original. DNA Replication Semiconservative Complementary Each new strand consists of one newly-synthesized strand and one strand inherited directly from the parent. This is known as being semi-conservative.

8 8 The model of replication was originally not well understood, so Understandings there were three different models that were hypothesized. U1: The replication of DNA is semiconservative and depends on complementary base pairing. Semi-conservative Conservative Dispersive DNA Replication Semiconservative Complementary

9 Skills S2: Analysis of Meselson and Stahl s results to obtain support for the theory of semiconservative replication of DNA. Meselson & Stahl Meselson and Stahl designed an experiment to determine which model was accurate. First, they grew a culture of bacteria in a broth containing N 15, a heavy isotope of Nitrogen. As the bacteria divided, they integrated the N 15 into their DNA, making it more dense. Then they transferred the bacteria to a broth with only N 14 9

10 Skills S2: Analysis of Meselson and Stahl s results to obtain support for the theory of semiconservative replication of DNA. After set periods of time, they centrifuged samples of the bacteria. This is technique used to used in an experiment to separate substances by density. They expected one of following outcomes: 10 Centrifuge

11 Skills S2: Analysis of Meselson and Stahl s results to obtain support for the theory of semiconservative replication of DNA. After the bacteria had time to duplicate, they removed samples and 11 separated their DNA using centrifugation. The results they obtained are shown here. Centrifuge

12 Skills S2: Analysis of Meselson and Stahl s results to obtain support for the theory of semiconservative replication of DNA. After one generation, the N 15 band had disappeared and a new band between N 15 and N 14 appeared. This indicated that new bacteria had 50% each of N 15 /N 14 in their DNA. 12 Centrifuge

13 Skills S2: Analysis of Meselson and Stahl s results to obtain support for the theory of semiconservative replication of DNA. In successive generations, an N 14 -only band appeared and grew darker while the N 15 / 14 band remained. 13 Centrifuge This indicated that new DNA consisted of one parental strand and one newly synthesized strand only containing N 14.

14 Skills S2: Analysis of Meselson and Stahl s results to obtain support for the theory of semiconservative replication of DNA. Meselson s and Stahl s observation in their experiments led them to conclude that the replication of DNA was a semi-conservative process and that the other models were incorrect. 14 Meselsohn & Stahl

15 Applications A1: Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR). In order to study genes and use them in experiments, large amounts must be obtained. A process called polymerase chain reaction (PCR) uses DNA polymerase to do this. 15 PCR

16 Applications A1: Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR). PCR In PCR reactions, all of the 16 components for DNA replication are added to test tubes, which are then put in thermo-cyclers Components include: Source DNA DNA Primers Free Nucleotides DNA Polymerase Reaction Buffer

17 Applications A1: Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR). DNA primers are used to isolate a gene. These are short strands of DNA that are complementary to regions on either side of the sequence of interest. 17 PCR

18 Applications A1: Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR). The process of PCR involves stages of cycling temperatures: Denuturing high temperature causes DNA strands to separate Annealing lower temperature allows DNA primers to bind 18 PCR Extending a medium temperature that is optimal for the Taq DNA polymerase enzyme. This allows for effective replication.

19 Applications A1: Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR). The enzyme used in PCR is Taq DNA polymerase, which is isolated from T. aquaticus. This bacteria is found in hot springs and so its enzymes have high optimum temperatures. 19 Taq DNA Polymerase Since PCR requires temperatures above body temperature, using this enzyme is much more efficient than human DNA polymerase.

20 Applications A1: Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR). Each cycle of PCR doubles the copies of the target gene. So, a large numbers of copies can be produced in a short period of time. 20 PCR

21 REVIEW 1. Discuss why DNA replication is important for organisms Outline the role of helicase and DNA polymerase in DNA replication. 3. Describe what is meant by the term semiconservative. 4. Analyze the findings of the Meselson-Stahl experiment. 5. Outline the use of Tag DNA Polymerase in PCR.

22 2.7 B Transcription

23 INTRO Before genes can be used to produce proteins, DNA must be converted into RNA. This is done through transcription. 23

24 Understandings U4: Transcription is the synthesis of mrna copied from the DNA base sequences by RNA polymerase. Transcripton is the process of creating mrna copies from DNA sequences. This is done by an enzyme called RNA polymerase. 24 Transcription RNA Polymerase To do this, a bubble of DNA is opened and used as a template. RNA polymerase then joins free-floating nucleotides complementary to the DNA strand.

25 25 Understandings U4: Transcription is the synthesis of mrna copied from the DNA base sequences by RNA polymerase. The RNA resulting from transciption has the same sequence as the target DNA sequence. However, there are two significant differences: RNA is singlestranded while DNA is doublestranded Transcription RNA contains uracil instead of thymine Uracil

26 Understandings S4: Deducing the DNA base sequence for the mrna strand. Determine the RNA code that would be produced by the following DNA sequences: 26

27 Understandings Determine the RNA code that would be produced by the following DNA sequences: 27 S4: Deducing the DNA base sequence for the mrna strand. U A A U U C C G G C U A U C C U A U C C G G U A A A U G C U A G U A C C C C G G U U A A U U C C U U A U G A

28 28 After transcription is complete, Understandings U4: Transcription is the synthesis of mrna copied from the DNA base sequences by RNA polymerase. Transcription mrna the resulting RNA is called messenger RNA (mrna). Replication and transcription occur in the nucleus, however proteins are made in the cytoplasm. So, the final mrna molecules leave the nucleus through holes called nuclear pores.

29 2.7 C Translation

30 Understandings U5: Translation is the synthesis of polypeptides on ribosomes. After transcription, mrna leaves the nucleus through nuclear pores. It travels to ribosomes, which are the cell structures responsible for synthesizing polypeptide chains. 30 Ribosomes are the site of translation. Translation

31 Understandings U5: Translation is the synthesis of polypeptides on ribosomes. During translation, ribosomes interpret the mrna sequence and synthesize polypeptide chains. The resulting proteins are typically released into the cytoplasm or rough ER. 31 Translation

32 Understandings U6: The amino acid sequence of polypeptides is determined by mrna according to the genetic code. U7: Codons of three bases on mrna correspond to one amino acid in a polypeptide. The sequence of amino acids in a polypeptide chain is determined by the sequence of the mrna nucleotides (A, U, C, G). 32 Codon Every three bases of mrna make up a codon. Each codon corresponds to an amino acid determined by the genetic code.

33 Understandings U6: The amino acid sequence of polypeptides is determined by mrna according to the genetic code. The genetic code is typically shown as a chart like the one below. 33 The bases of the codon correspond to an amino acid or a stop signal. Some AA s have multiple codons, while others only have one. U7: Codons of three bases on mrna correspond to one amino acid in a polypeptide. Genetic Code

34 Understandings U8: Translation depends on complementary base pairing between codons on mrna and anticodons on trna. trna Codon During translation, the ribosome reads each mrna codon and matches it with a trna molecule. Each trna has two distinct sites: 1. An anticodon, which is a three-base sequence complementary to the codon. 2. An amino acid that will be added to the polypeptide chain. 34

35 Understandings U8: Translation depends on complementary base pairing between codons on mrna and anticodons on trna. As the ribosome reads a mrna strand, the AA s on the trna s are bound via condensation to form a polypeptide chain. 35

36 Skills S1: Use a table of the genetic code to deduce which codon(s) corresponds to which amino acid. Using an Amino Acid Table 1. Identify the three bases of the codon. 2. Find the first base on the left-hand side. 3. Find the second base on the top and identify the square where they intersect. 4. Find the third base on the right hand side and identify the amino acid for the full codon. 36

37 37 Skills S1: Use a table of the genetic code to deduce which codon(s) corresponds to which amino acid. Using an Amino Acid Table Use the chart to determine what AA corresponds to: - UAC - AAG - CUG - GAU - UAA

38 38 Skills S1: Use a table of the genetic code to deduce which codon(s) corresponds to which amino acid. Using an Amino Acid Table Use the chart to determine what codons correspond to: - Serine - Histidine - Valine - Arginine

39 Skills S1: Use a table of the genetic code to deduce which codon(s) corresponds to which amino acid. Using the chart, determine what codons correspond to: Serine: Histidine: Valine: Arginine: - UCU, UCC, UCA, UCG, AGU, AGC - CAU, CAC - GUU, GUC, GUA, GUG 39 - CGU, CGC, CGA, CGG, AGA, AGG

40 Skills S3: Use a table of mrna codons and their corresponding amino acids to deduce the sequence of amino acids coded by a short mrna strand of known base sequence. Use an amino acid chart to determine the AA sequence of the following mrna chains. 40

41 Skills S3: Use a table of mrna codons and their corresponding amino acids to deduce the sequence of amino acids coded by a short mrna strand of known base sequence. Use an amino acid chart to determine the AA sequence of the following mrna chains. Met Leu Gly Lys Gln Stop Met Phe Lusc Ala Glu Stop 41 Met Arg Ile Phe Arg Stop

42 Applications A2: Production of human insulin in bacteria as an example of the universality of the genetic code allowing gene transfer between species. Insulin The same genetic code is used by all organisms. So, the same gene in one organism will produce the same protein in others. Humans take advantage of this in the production of insulin. The human gene is inserted into bacteria, which are then able to produce insulin. Large amounts of insulinproducing bacteria are grown and then the hormone is harvested for human use. 42

43 REVIEW 1. Define translation Outline how the ribosome reads mrna and synthesis polypeptide chains. 3. Outline the structure of trna molecules. 4. Outline the steps needed to interpret mrna with an amino acid chart. 5. Explain the universality of the genetic code using insulin as an example.