1. DNA replication, Transcription & Translation
Subtopic 2.7 | Mrs. Palmer | BHS

2. The essential Idea: 
Genetic information in DNA can be accurately copied and translated to make proteins needed by the cell.
Objectives:
Learn how DNA is replicated and how genes direct the synthesis of proteins.
How cells make protein from DNA.

3. Review 1) Which phase of cell division replication of DNA takes place?
2) What are genes made of?
3) What do we mean when we say that DNA stores genetic information? What kind of information are we referring to?
4) Where is the genetic information found for eukaryotes?
5) where is the genetic information found for prokaryotes?

4. Protein Synthesis happens in 2 steps:
Transcription
transcribing DNA through the synthesis of messenger RNA (mRNA) copied from the DNA base sequences.
Translation
translating the genetic code through the synthesis of proteins (more specifically polypeptide chains) on ribosomes.

5. Meselson and Stahl’s DNA replication experiment.
Provided evidence for the theory of semi-conservative replication of DNA.

6. Meselson and Stahl’s DNA replication experiment.
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7. Data-based questions: The Meselson and Stahl experiment.
1. DNA was produced containing 14N, rather than 15N in the organic bases; 14N has a lower mass than 15N;
2. a) g cm-3 b) falsifies conservative replication because that method would give two bands of DNA with densities of and g cm-3 ; dispersive unlikely to give a band half way between the higher and lower densities;
3. a) two bands; density and g cm-3 ; equal amounts of the two bands; b) falsifies the dispersive mechanism; there would only be one band; all the DNA would be partly and partly g cm-3 ;
4. less and less g cm-3 DNA; because all new strands are g cm-3 ; and when these strands are replicated the DNA produced is g cm-3 ;
5. semi-conservative redrawn; next generation has two red-green molecules and two all green ones; generation after has two red-green molecules and six all green ones;
6. three bands with 1.710, and g cm-3 density;

8. 2.7.1 DNA replication
Copying DNA is called replication and that happens during the S phase of the cell cycle.
In most cases, replication results in identical copies of the DNA in the daughter cells. 
Mutations in the DNA, or errors when the DNA is copied, can cause tumorogenesis.

9. Replication can be divided into separate steps.
First step: Unwind the coils to make the strands accessible to enzymes.
This happens first because DNA is normally super coiled by being tightly wound around nucleosomes.
The enzyme helicase then unwinds the double helix and separates the two DNA strands by breaking the hydrogen bonds between the bases. 
Second step: DNA polymerase can start making new strands of DNA by using the two 'old parent' strands as templates.

10. BEWARE
As the two DNA strands of the double helix are anti-parallel to each other, DNA polymerase proceeds in opposite directions during replication. On one strand it moves in the same direction as the replication fork (immediately behind helicase enzyme), and in the reverse direction on the other strand.
DNA replication always occurs in 5' to 3' direction.

11. Important
DNA replication is semi-conservative as each daughter molecule formed contain one original strand from the parent molecule and one newly synthesized strand.

12. 2.7.2 DNA transcription
Eukaryotic DNA is contained within the nucleus.
Protein Synthesis takes place in ribosomes.
Transcript is needed to bring the information to the ribosome where protein are produced.
Transcription definition = a written or printed version of material originally presented in another medium.

13. 2.7.2 DNA transcription
The coding information is transcribed into a special molecule called messenger RNA (mRNA).
The DNA functions as a template, and the single-stranded mRNA molecule that is made follows the complementary base pairing rules of DNA - with one exception. 
In DNA G(uanine) always pairs with C(ytosine) and A(denine) pairs with T(hymine).
In RNA U(racil) replaces T(hymine)
so in an mRNA molecule, whenever there is an A in the DNA template, a U will appear in the newly formed mRNA.

14. What is the mRNA made by this strand?
GCU CCU GAG UUG

15. The role of RNA Polymerase
A gene is a section of DNA on a chromosome that codes for a particular protein.
In transcription, the section of DNA that contains the required gene is unwound and separated so that RNA polymerase enzymes can access the DNA bases. 
The RNA polymerase then transcribes a sequence of DNA bases into mRNA.
RNA polymerase is responsible for both separating the DNA strands of the double helix as well as for joining the ribonucleotides together by phosphodiester bonds to form an mRNA strand.

16. Transcription
The DNA strand that is not transcribed is called the sense strand and has the same sequence of bases as the mRNA molecule, except for thymine being replaced by uracil.
The transcribed strand is known as the antisense strand and is complementary to the mRNA molecule.

17. BEWARE
Each chromosome contains many genes, however only a few of these are expressed at any given time - these are the ones that are transcribed.

18. 2.7.3 DNA translation and the genetic code
Translation is the synthesis of polypeptides on ribosomes.
Each codon of three bases on the mRNA is translated into one amino acid in a polypeptide chain.
The sequence of the codons on the mRNA determines the amino acid sequence of the polypeptide made. Since the DNA of the gene directs the synthesis of mRNA, any changes affecting the base sequence of the DNA may lead to the integration of the wrong amino acid/s in the polypeptide made.

19. The genetic code is DEGENERATE
This means that some amino acids that are encoded by more than one codon.
There is a total of 20 amino acids, however, 64 codons can be formed by using the 4 bases. Therefore, some amino acids are coded for by more than one codon accounting for the degeneracy of the genetic code. There are also specific codons which signal the protein translation machinery to start or to stop.

20. The genetic code is universal
This means that the genetic information in bacteria is translated in the same way as that of elephants, sequoia  trees, or any other living organism.
The table is read from the left to the right (first base, then second, and finally, third base).

22. Protein Size
The length of the mRNA depends on the size of the gene and is directly related to the size of the protein that is made.
For example, if a mRNA is 456 nucleotides in length, it can code for a protein that is 151 amino acids long.
The explanation for this is that three nucleotides are needed for the start codon, which codes for the amino acid methionine. Three nucleotides are needed for the stop codon, which leaves 450 nucleotides. Divide by 3 (each set of three nucleotides form one codon) and you get 150 codons = 150 amino acids, so the total length of the protein is 151 amino acids (when you add the amino acid methionine).

23. Translation The translation takes place in the cytoplasm of the cell.
A transfer RNA (tRNA) molecule brings a specific amino acid to the mRNA. tRNA molecules have an anticodon that pairs with a codon of the mRNA, ensuring that the correct amino acid is added to the growing polypeptide chain.
Common complementary base pairing rules define this interaction.

24. tRNA
RNA molecule that transfers amino acids over to the ribosome where protein synthesis take place.
The following diagram shows the schematic form of a tRNA molecule with its anticodon, and the 3' end, where the amino acid attaches itself.

25. Translation
In mammalian cells, the average length of a mRNA is about 2,000 nucleotides, which translates roughly to a protein of around 650 amino acids in length.

26. Ribosome translocation

27. Review DNA ----> mRNA ----> protein.
The order of nucleotides defines the order of codons, which defines the order of amino acids, which is the primary structure of a protein.
tRNA (anticodon) pairs with mRNA (codon).
Translation occurs in 5' to 3' direction.
All interactions between DNA and RNA, and RNA and RNA follow complementary base paring rules.
Remember DNA - A pairs with T, and C pairs with G.
RNA - A pairs with U, and C pairs with G.
The genetic code is universal!

28. 2.7.4 PCR Polymerase Chain Reaction.
The discovery of this technique has revolutionized medical science, forensic science and molecular biology. PCR has enabled scientists to clone genes, to work with minute amounts of DNA found at crime scenes, identify the dead and, perhaps most extraordinarily, sequence the DNA of extinct humans and other life forms.
The PCR technique for amplifying DNA was developed by Kary Mullis in 1983, earning him a Nobel prize for his work. 
PCR is a technique that can make billions of copies of one molecule of DNA by repeatedly copying a specific stretch of that DNA. The technique uses the cyclic heating and cooling of a DNA sample in the presence of primers, DNA nucleotides and Taq polymerase to amplify the DNA.

29. PCR

30. PCR video 2

31. PCR continued
Taq polymerase is a DNA polymerase isolated from a bacterium, Thermus aquaticus.  It lives in hot water springs at temperatures between 50oC and 80oC. Most of its proteins are thermostable, which means they can operate at higher temperatures than other organisms. As PCR uses high temperatures, it requires a special DNA polymerase that can withstand these higher temperatures.

32. 2.7.5 Insulin production
You should know by now that the genetic code is universal. A gene for a human protein is translated by using the same codons as a bacterial gene, thus opening up endless possibilities. 
Placing a gene from one organism into a different organism results in a transgenic organism. Microorganisms, such as bacteria or yeast, are made transgenically by inserting a gene from another organism into the cell. The organism then becomes a biofactory for many of today's therapeutic drugs.
People suffering from diabetes require insulin to treat their disease. Insulin is a hormone produced by the beta cells of the pancreas. It regulates glucose uptake and conversion of glucose to glycogen in the liver.
Insulin used to be extracted from the pancreas of pigs or cattle because the structure of this hormone in those animals is very similar to that of human insulin. But the purification process was not very efficient and some patients developed allergic reactions to the animal insulin. In 1982, the human gene for insulin was transferred to the E. coli bacterium, resulting in the production of human insulin. Now diabetics can be treated with human insulin, which causes fewer problems than insulin extracted from other species.

33. Insulin production
The production of human insulin in bacteria is a reality today due to the universality of the genetic code which allows gene transfer between species.