1. Chapter 12: Molecular Biology of the Gene (Outline)
DNA Structure
Watson and Crick Model
DNA Replication
Semiconservative Replication
Prokaryotic versus Eukaryotic Replication
Types of RNA
Gene Expression
The Genetic Code
Transcription
Translation

2. Structure of DNA DNA contains Each nucleotide consists of
Two Nucleotides with purine bases (double ring)
Adenine (A)
Guanine (G)
Two Nucleotides with pyrimidine bases (single ring)
Thymine (T)
Cytosine (C)
Each nucleotide consists of
Deoxyribose (5-carbon sugar)
Phosphate group
A nitrogen-containing base

3. Chargaff’s Rules
In 1947, Erwin Chargaff had developed a series of rules based on a survey of DNA composition in organisms
The amounts of A, T, G, and C in DNA varies from species to species
In each species, the amount of A=T and the amount of G=C
All this suggests DNA uses complementary base pairing to store genetic info

5. Rosalind Franklin’s Work
Was an expert in X-ray crystallography
Technique used to examine DNA fibers (under right conditions form a crystal)
Concluded that DNA is a double helix

6. Watson and Crick Model Watson and Crick, 1953
Constructed a model of DNA from Franklin’s X-ray diffraction
Double-helix model is similar to a twisted ladder
Sugar-phosphate backbones make up the sides
Hydrogen-bonded bases make up the rungs ‘steps’
Model agreed with Chargaff’s rule or complementary base pairing
Received a Nobel Prize in 1962

7. Watson/Crick Model of DNA

8. Watson and Crick Model (cont.)
Antiparallel nature: the sugar- phosphate backbone of each strand runs in opposite directions
One strand runs 5’ to 3’, while the other runs 3’ to 5’
The nucleotides connect at the hydroxyl group of the 5 carbon sugar (at the 3’ end)
DNA strand is made in a 5’ to 3’ direction

9. Replication of DNA
DNA replication: the process of copying a DNA molecule
Each old DNA strand serves as a template
Replication involves 3 main steps
Unwinding – original double helix strands (parental DNA) are unwound and “unziped” by helicase enzyme
Complementary base pairing – positioning of new complementary nucleotides
Joining – complementary nucleotides join to form new strands
Each daughter DNA contains an old & new strand

10. Semiconservative Replication of DNA
DNA polymerase: enzyme complex that carries out the last two steps in DNA synthesis
DNA replication must occur before cellular division
Cancer cells are treated with chemotherapeutic drugs “analogs”, which causes replication to stop and cells to die off

11. Replication: Prokaryotic vs. Eukaryotic
Prokaryotic Replication
Bacteria have a single circular loop of DNA
Replication moves around the circular DNA molecule in both directions
The process begins at the origin of replication and always occur in the 5’ to 3’ direction
Replication stops when the 2 DNA polymerases meet at a termination region
Bacterial cells require about 40 min to replicate the complete chromosome

12. Prokaryotic DNA Replication

13. Replication: Prokaryotic vs. Eukaryotic
Eukaryotic Replication
DNA replication begins at numerous points (origins of replication) along linear chromosome
DNA unwinds and unzips into two strands through the action of helicase enzyme
Each old strand of DNA serves as a template for a new strand
Replication bubbles spread bi-directionally until they meet
Replication fork – V shape formed during DNA replication

14. Eukaryotic DNA Replication

15. Eukaryotic DNA Replication (cont.)
Eukaryotes replicate their DNA at a slower rate 500–5,000 base pair per minute
Eukaryotic cells, however complete DNA replication in a matter of hours, how?
The linear chromosomes also pose a problem:
DNA polymerase cannot replicate the ends of chromosomes that contain telomeres (short segments of DNA repeated over and over)
Instead, telomerase enzymes add the repeats after chromosome replication
In stem cells, this process preserves the ends of chromosomes and prevents the loss of DNA

16. Accuracy of Replication
DNA polymerase is very accurate with approx. one mistake per 100,000 base pairs
DNA polymerase is also capable of proof reading the daughter strand
It recognizes a mismatched nucleotide and removes it from a daughter strand, how?
By reversing direction and removing several nucleotides
After removing the mismatched nucleotide, it changes direction again and continues
Overall, the error rate for the bacterial DNA polymerase is only one in 100 million base pairs!

17. The Genetic Code of Life
The mechanism of gene expression
Gene – segment of DNA that specify information, but information is not structure and function (i.e. protein)
Genetic info is expressed into structure and function through protein synthesis
DNA in gene controls the sequence of nucleotides in an RNA molecule
RNA controls the primary structure of a protein

18. RNA Carries the Information
Like DNA, RNA is a polymer of nucleotides
RNA nucleotides are of four types: U, A, C & G
Uracil (U) replaces thymine (T) of DNA
There are three major classes of RNA
Messenger RNA (mRNA) - takes a message from DNA in the nucleus to ribosomes in cytoplasm
Transfer RNA (tRNA) – transfers amino acids to the ribosomes
Ribosomal RNA (rRNA) – and proteins make up ribosomes which read the message in mRNA

19. Structure of RNA

20. The Genetic Code
The central dogma of molecular biology states that the flow of genetic information is “DNA to RNA to protein”
There must be a genetic code for each of the 20 amino acids found in proteins
However, can four nucleotides provide enough combinations to code for 20 amino acids?
The genetic code is a triplet code, comprised of three-base code words (e.g. AUG).
A codon consists of 3 nucleotide bases of DNA, why?

21. Central Dogma in Molecular Biology
Transcription: DNA serves as a template for RNA formation
Translation: mRNA transcript directs the amino acid sequence in a polypeptide

22. Finding the Genetic Code
Nirenberg and Matthei (1961) found that an enzyme that could be used to construct a synthetic RNA in a cell-free system; they showed the codon UUU coded for phenylalanine
By translating just three nucleotides at a time, they assigned an amino acid to each of the RNA codons and discovered important properties of the genetic code

23. Properties of the Genetic Code
The code is degenerate
There are 64 codons available for 20 amino acids
Most amino acids encoded by two or more codons (e.g. luecine and serine), why?
The genetic code is unambiguous
Each triplet codon specifies one and only one amino acid
The code has start and stop signals
There are one start codon and three stop codons (sequences)

24. The Code is Universal
With few exceptions, all organisms use the code the same way
Genetic code used by mammalian mitochondria and chloroplasts differs slightly
The universal nature of the genetic code suggests the code dates back to the very first organisms and that all organisms are related
It is possible to transfer genes from one organism to another – genetic engineering
Example: Glowing mice

25. mRNA Codons

26. Steps in Gene Expression: (1) Transcription
Messenger RNA is formed
A DNA segment helix unwinds and unzips, thus serving as a template for mRNA formation
Loose RNA nucleotides bind to exposed DNA bases using the C=G AND A=U rule
The information is in the base sequence of the “template” strand of the DNA molecule
RNA polymerase connects the loose RNA nucleotides together in the 5’ → 3’ direction

27. Transcription of mRNA
Transcription (initiation) begins when RNA polymerase attaches to a promoter on DNA
Promoter – region of DNA which defines the start of the gene, the direction of transcription, and the strand to be transcribed
The RNA-DNA association is not as stable as the DNA double helix
Only the newest portion of the RNA molecule with RNA polymerase is bound to DNA; the rest dangles off to the side

28. Transcription of mRNA (cont.)
Elongation of mRNA continues until RNA polymerase comes to a DNA stop sequence
Results in the release the mRNA transcript
Many RNA polymerase molecules work to produce mRNA from the same DNA region at the same time
Either strand of DNA can be a template strand but for a different gene

29. Transcription

30. Steps in Gene Expression: (2) Translation
Translation takes place in the cytoplasm of eukaryotic cells
Translation is the second step by which gene expression leads to protein synthesis
The sequence of codons in the mRNA at a ribosome directs the sequence of amino acids in a polypeptide
One language (nucleic acids) is translated into another language (protein)

31. The Role of Transfer RNA
The tRNA molecule transfers amino acids to the ribosomes
The amino acid binds to the 3’ end; the opposite end of the molecule contains an anticodon that binds to the mRNA codon in a complementary fashion
There is at least one tRNA molecule for each of the 20 amino acids found in proteins
There are fewer tRNAs (40) than codons (61) as some tRNAs pair with more than one codon

32. Structure of tRNA

33. Translation Requires Three Steps
During translation, mRNA codons base-pair with tRNA anticodons carrying specific amino acids
Codon order determines the order of tRNA molecules and the sequence of amino acids in polypeptides
Protein synthesis involves 3 steps: initiation, elongation, and termination
Enzymes are required for all three steps; energy (ATP) is needed for the first two steps

34. Steps in Translation: 1. Initiation
Components necessary for initiation are
Small ribosomal subunit
mRNA transcript
Initiator tRNA, and
Large ribosomal subunit
Initiation factors - special proteins that bring the above together
Initiator tRNA
Always has the UAC anticodon
Always carries the amino acid methionine
Capable of binding to the P site of ribosome

35. Steps in Translation: 1. Initiation (cont.)
Chain Initiation
In prokaryotes, a small ribosomal subunit attaches to mRNA at the start codon (AUG)
Initiator tRNA (UAC) pairs with this codon; then the large ribosomal subunit joins to the small subunit
Each ribosome contains three binding sites – the P site (for peptide), the A site (for amino acid), and the E site (for exit)
The initiator tRNA binds to the P site
The A site is for the next tRNA carrying the next aa
The E site is to discharge tRNAs from the ribosome

36. Initiation

37. Steps in Translation: 2. Elongation
Elongation – refers to the growth in length of the polypeptide one amino acid at a time
The tRNA with attached polypeptide is at the P site; a tRNA-amino acid complex arrives at the A site
Elongation factors – proteins that facilitate complementary base pairing between the tRNA anticodon and the mRNA codon at the ribosome
The polypeptide is transferred and attached by a peptide bond to the newly arrived amino acid in the A site via a ribozyme and energy (ATP)

38. Steps in Translation: 2. Elongation (cont.)
The tRNA molecule in the P site is now empty
Translocation occurs with mRNA, along with peptide-bearing tRNA, moving to the P site and the spent tRNA moves from the P site to the E site → exits the ribosome
As the ribosome moves forward three nucleotides, there is a new codon now located at the empty A site
The complete cycle is rapidly repeated, about 15 times per second in the bacterium E. coli

39. Elongation

40. Steps in Translation: 3. Termination
Termination of polypeptide synthesis occurs at a stop codon
UAA, UAG, or UGA
Does not code for an amino acid
The polypeptide is enzymatically cleaved from the last tRNA by a release factor
The tRNA and polypeptide leave the ribosome, which dissociates into its two subunits
The released polypeptide begins to take on its 3D shape

41. Termination