1. Today: 1. DNA. Replication 2. Protein Synthesis 3. DNA Structure
Today: DNA Replication 2. Protein Synthesis 3. DNA Structure Activity

2. The Structure of a DNA Molecule:

3. Replicating DNA…
Model??

4. PURINES
PYRIMIDINES
Pairing of PURINES with PYRIMIDINES is key to both Structure and Replication of DNA

5. One more important limitation for our model of DNA replication:
DNA Molecules are ANTI-PARALLEL
5’3’ = FORWARD STRAND
3’5’ = REVERSE STRAND
DNA Polymerases can only add bases to the 3’ end of a DNA Molecule!

6. The three models of DNA Replication: CONSERVATIVE, SEMICONSERVATIVE and DISPERSIVE.

7. This model allows each strand to serve as a TEMPLATE for synthesis of a new strand.
Watson and Crick’s model is a SEMICONSERVATIVE MODEL (each daughter molecule will have one “parent” strand and one newly synthesized strand)

8. An Overview of the Mechanics of DNA Replication:
Must be FAST and ACCURATE (~1 mistake/billion bases)!

9. High Energy Covalent Bonds!
With the two strands separated in a REPLICATION BUBBLE, a DNA Polymerase can add dNTPs (deoxyribose “N” triphosphate)
COUPLED REACTION: hydrolysis of pyrophosphate is exergonic

10. One more important limitation for our model of DNA replication:
DNA Molecules are ANTI-PARALLEL
5’3’ = FORWARD STRAND
3’5’ = REVERSE STRAND
DNA Polymerases can only add bases to the 3’ end of a DNA Molecule!

11. The LEADING STRAND is a CONTINUOUS molecule.
Because DNA Polymerase can only attach nucleotides to the 3’ end of a growing DNA molecule, the two strands of Parental DNA are replicated differently.
The LEADING STRAND is a CONTINUOUS molecule.
The LAGGING STRAND is composed of OKAZAKI FRAGMENTS “stitched” together by DNA LIGASE.

12. Other Key Players:
Another enzyme, HELICASE, untwists the double helix while SINGLE-STRAND BINDING PROTEINS hold them apart so that DNA Replication can proceed.

13. One more Complication:
DNA POLYMERASE can’t initiate DNA Replication! It can only add nucleotides to an existing chain.
Thus DNA Replication requires a PRIMER, and an enzyme, PRIMASE, to make it!

14. Let’s Watch!

16. DNA Replication: A more Complete View

17. Catching & Repairing Mistakes
DNA Polymerases err at a rate of 1/10,000 base pairs.
“Proofreading” reduces the error rate to 1/1 billion base pairs.
Cells can repair many errors; Humans have 130 known DNA repair enzymes!

18. The Importance of DNA Repair
The ability to repair damaged DNA is critical to long-term survival.
Individuals with xeroderma pigmentosum produce defective nucleotide excision repair enzymes.
Without these repair enzymes, mutated DNA in skin cells (UV) is not repaired, leading to skin cancers.

19. Another Challenge: The End Replication Problem
As eukaryotes replicate their DNA, they are unable to “fill-in” the 5’ end of new molecules.
Thus, over time, the daughter molecules become shorter and shorter.

20. The Solution:
Telomeres- noncoding multiple repeats of a short nucelotide sequence (TTAGGG)
Protects genes (coding sequences) from being eroded. (Erosion triggers programmed cell death!)
Telomeres are regenerated by telomerases in germ-line cells and most cancers.

21. Was Dolly old before her time?
Dolly the Sheep may have been susceptible to premature ageing, new research suggests. A team from the biotechnology firm PPL Therapeutics in Scotland examined Dolly's telomeres.
They report in the journal Nature that the structures are slightly shorter than would be expected in a sheep of her age born normally.
Source: BBC NewsThursday, May 27, 1999

22. A Thought Exercise:
Work with your neighbor(s) to diagram normal DNA replication. Then, come up with a list of INGREDIENTS and CONDITIONS you would need to replicate DNA in a test tube!

23. The Polymerase Chain Reaction (PCR)
DNA Replication in a tube!
Exponential process used to generate large quantities of specific regions of DNA for use in forensics, DNA sequencing, research, medical diagnostics, etc.

24. Let’s Watch This, Too…

25. A Challenge Question For You…
Examine the structure of a common anti-HIV drug, AZT. Compare its structure to that of a normal nucleotide, deoxy-thymidine (T; top). Based on your comparison of these two structures, suggest a mechanism to explain the ability of AZT to reduce the viral load of an HIV+ patient.

26. Next Up: A Missing Link!

27. Getting From a Gene to a Polypeptide Occurs in 2 Steps:
TRANSCRIPTION:
transcribing DNA into RNA
2. TRANSLATION:
translating RNA into an amino acid sequence ?

28. What differences do you notice??

29. Prokaryotic Transcription & Translation
In Prokaryotes, transcription and translation can occur simultaneously.

30. Eukaryotic Transcription & Translation
In Eukaryotes, transcription and translation are separated. Transcription occurs in the nucleus, generates a primary transcript (pre mRNA) which is edited and exported as mRNA.

31. In order to translate DNA (RNA) you must first crack the genetic “code”!
The genetic code is a triplet code in which a group of three bases (codon) of a DNA molecule code for a particular amino acid.

32. Reading the Genetic Code:
Note that the code is sometimes redundant, but never ambiguous.
It is also (nearly) universal, allowing for DNA from one species to function in another! (transgenic organisms)

33. Arabidopsis thaliana C24 wild type (left) and transformed (GFP; right)
Tobacco plant expressing a firefly gene (luciferase)
Herman, at right, is the first transgenic dairy animal engineered to make the human milk protein, lactoferrin, which is an antibacterial protein that can be used to treat immunosuppressed patients and could be incorporated into infant formula.

34. TRANSCRIPTION occurs in 3 Steps:
Initiation
Elongation, and
Termination

35. Initiation of Transcription:
Important Details!

36. Step 2: Elongation
Once the RNA polymerase is attached to the promoter DNA, the DNA strands unwind, and the RNA Polymerase begins to TRANSCRIBE the template strand.

37. Step 3: Termination
Transcription continues until AFTER the RNA polymerase transcribes a TERMINATOR SEQUENCE. This transcribed terminator (RNA) acts as the signal!

38. Step 3: Termination
In prokaryotes, transcription generally stops right at the end of the termination signal, with the polymerase releasing both the RNA and DNA molecules.

39. Step 3: Termination
In eukaryotes, the polymerase may continue for thousands of bases past the termination signal (AAUAAA). ~10-15 bases past this termination signal the pre-mRNA is cut free from the enzyme for RNA PROCESSING
Let’s Watch!

40. RNA Processing: 1. Modifying the ends of the pre-mRNA
2. Splicing the interior of the pre-mRNA
At the 3’ end, and enzyme attaches and creates a poly(A) tail ( adenine nucleotides). This 3’ poly(A) sequence:
1. inhibits degradation
2. assists with ribosome binding
3. facilitates the export of the mRNA from the nucleus.
The 5’ end is immediately “capped” with a modified guanine (G). This 5’ cap:
1. protects the molecule from degradation
2. is recognized by the ribosome to initiate translation

41. ASIDE: Understanding RNA processing provides enormous advantages for applying Genetic Technology!
Example: RT-PCR
Extract RNA, not DNA

42. RNA Processing: Step 2. Splicing the interior of the pre-mRNA
Eukaryotic genes contain INTRONS (long noncoding regions of nucleotides that are not translated). An average transcription unit is ~8000 nucleotides, but only ~1200 nucleotides are used to code for an average protein.
(**How many amino acids in an average protein?)

43. The Mechanism for removing INTRONS
Short nucleotide sequences at the ends of introns act as signals for splicing.
Small nuclear ribonucleoproteins (snRNPs) recognize these sites.
Several snRNPS combine with additional proteins to form a SPLICEOSOME.
The SPLICEOSOME cuts at specific points, and joins the ends of the two exons together.

44. Why Introns??

45. Possible Functions/Benefits of RNA Splicing:
Regulatory Role? (Transcriptional regulation of gene expression)
Alternative RNA splicing (make many different proteins from the same “gene”)
Facilitate the evolution of new proteins (DOMAINS may be exchanged during crossover. Introns provide more places for crossover to occur.)

46. From mRNA to Protein
Edited mRNA is then exported from the nucleus to the cytoplasm where the protein will be assembled

47. Translation: From mRNA to Protein
mRNA is translated by an interpreter, transfer RNA (tRNA).
tRNA transfers the correct amino acid from the cytoplasm to a ribosome.
The ribosome adds amino acids to the growing end of a polypeptide chain.

48. tRNA:
Each type of tRNA molecule carries a specific amino acid, and contains a specific anticodon.
The anticodon is a nucleotide triplet that binds with its complementary codon on mRNA

49. tRNA’s:
Note the directional nature of this process! mRNA’s are typically read 5’  3’. Therefore anticodons are 3’ 5’.

50. For this system to work, the correct amino acid must be attached only to tRNA molecules carrying the correct anticodon.
Correct attachment is catalyzed by one of 20 different Aminoactyl-tRNA synthetase enzymes.
Endergonic? Exergonic?

51. Ribosome Structure:
Ribosomes are made of two subunits (the large and small). Each subunit is made of proteins and ribosomal RNA (rRNA).
Eukaryotes assemble their ribosomes in the nucleus (importing the protein component from the cytoplasm). The subunits are then exported to the cytoplasm, where they assemble upon binding a mRNA molecule.
Mouse liver

52. (ex. Tetracycline, streptomycin)
Differences in Eukaryotic and Prokaryotic Ribosome Structure Make Good Drug Targets!
(ex. Tetracycline, streptomycin)

53. 3 binding sites for tRNA:
P site- holds the tRNA carrying the growing polypeptide chain
A site- holds the tRNA carrying the next amino acid
3. E site- where the discharged tRNAs leave the ribosome.
(The ribosome is an ENZYME that catalyzes the formation of a peptide bond between amino acids!)

54. TRANSLATION also occurs in 3 steps:
Initiation- the RIBOSOME assembles on the mRNA molecule at the START CODON
Elongation- the polypeptide chain is assembled sequentially as amino acids are covalently bound to one another
Termination- the Ribosome reaches a STOP CODON and releases the mRNA and Polypeptide

55. Initiation- The small ribosomal subunit binds to a mRNA
An initiator tRNA (UAC) pairs with the start codon (AUG)
The large ribosomal subunit binds to complete the Translation Initiation Complex.
Initiation, Elongation, and Termination
Initiation requires energy and initiation factors

56. Step 2: Elongation Elongation Factor
Requires energy! mRNA moves through the ribosome

57. Amino Acid Structure and the Formation of Polypeptides
A quick review:
Amino Acid Structure and the Formation of Polypeptides
Note the Amino and Carboxyl ends. (Also called the N-terminus and the C-terminus)

58. Step 3: Termination Let’s Try!
Elongation continues until a stop codon in the mRNA reaches site A. A RELEASE FACTOR protein binds to the stop codon in the A site, causing the completed polypeptide to be released from the ribosome.
Let’s Try!

59. Many single ribosomes can translate a given mRNA molecule simultaneously (POLYRIBOSOMES).

60. Post-Translational Modification
Sugars, lipids, phosphate groups, etc. may be added to amino acids in the polypeptide
Amino acids may be cleaved (removed) from the polypeptide, or the entire chain may be cut into multiple pieces

61. Getting Proteins Where They’re Going: Signal Peptides
All ribosomes begin assembling proteins in the cytoplasm (free ribosomes).
If a signal peptide is present, they bind to the ER (bound ribosomes)

62. Getting Proteins Where They’re Going: Signal Peptides

63. Understanding Transcription and Translation Allows You to Predict the Effect of Mutations!

64. Understanding Gene Mutations
1. Base-pair SUBSTITUTIONS-
What happens if UCA UCC ?

65. Understanding Gene Mutations
1. Base-pair SUBSTITUTIONS-
What happens if UAC UAG?

66. Sickle Cell Disease: a Substitution

67. The Effects:

68. Understanding Gene Mutations
2. INSERTIONS and DELETIONS
One+ base pairs are inserted or deleted from a DNA sequence.
Insertions and Deletions frequently result in FRAMESHIFT MUTATIONS.
Example: CCR5 and HIV/Plague Resistance!

69. Insertions or Deletions Can Result in Missense or Nonsense