1. Polypeptide Synthesis

2. How does the DNA control the cell’s functions?
DNA makes proteins which dictate the function of the cells
ONE GENE ONE ENZYME - Beadle and Tatum concluded that each step in the metabolism of arginine is controlled by a separate enzyme and that each enzyme is controlled by a specific gene - therefore a change in the gene would affect the enzyme and therefore affect the metabolism of the individual
One Gene One Enzyme meant that each gene was responsible for the production of one enzyme

3. (All necessary nutrients)
(Missing key nutrients)

4. EXPERIMENT RESULTS Class I Mutants Class II Class III Wild type
Minimal
medium
(MM)
(control)
MM +
Ornithine
Citrulline
Arginine
Working with the mold Neurospora crassa, George Beadle and Edward Tatum had isolated mutants requiring arginine in their growth medium and had shown genetically that these mutants fell into three classes, each defective in a different gene. From other considerations, they suspected that the metabolic pathway of arginine biosynthesis included the precursors ornithine and citrulline. Their most famous experiment, shown here, tested both their one gene–one enzyme hypothesis and their postulated arginine pathway. In this experiment, they grew their three classes of mutants under the four different conditions shown in the Results section below.
The wild-type strain required only the minimal medium for growth. The three classes of mutants had different growth requirements

5. CONCLUSION
From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the one gene–one enzyme hypothesis and also confirmed the arginine pathway.
(Notice that a mutant can grow only if supplied with a compound made after the defective step.)
Class I
Mutants
(mutation
in gene A)
Class II
in gene B)
Class III
in gene C)
Wild type
Gene A
Gene B
Gene C
Precursor
Ornithine
Citrulline
Arginine
Enzyme A B C

6. CLARIFICATION not all proteins are enzymes
therefore ONE GENE ONE PROTEIN
however, not all proteins are made of one polypeptide - some are conglomerates (quaternary structure) of different kinds of amino acid chains - each chain being made by a different gene
therefore ONE GENE ONE POLYPEPTIDE

7. From Gene to Protein Three Basic Steps:
1. Transcription - DNA to RNA - RNA copied from DNA
Participants: DNA (Promoter region, TATA box, Transcription Unit, Terminator region), Transcription Factors, RNA polymerase, RNA nucleotides
2. RNA Processing
Participants: Pre-RNA (Introns and Exons), SNRNPs, GTP, Adenines
3.Translation: RNA to Protein
Participants: m-RNA, t-RNA, amino-acyl t-RNA synthetase, ribosomes, Amino Acids, Release factor

8. Figure 17.4 Gene 1 Gene 2 Gene 3 DNA strand (template) mRNA Protein
molecule
Gene 1
Gene 2
Gene 3
DNA strand
(template)
TRANSCRIPTION
mRNA
Protein
TRANSLATION
Amino acid A C G T U
Trp
Phe
Gly
Ser
Codon 3 5

9. TRANSCRIPTION process where one side of the DNA is copied into RNA
Template Strand - side of the DNA copied (3’5’)
section of DNA transcribed is called the transcription unit
Differences in DNA and RNA
deoxyribose : ribose
Thymine: Uracil
2 strands: 1 strand

10. Composition of DNA to be Transcribed
1. Promoter region:
- TATA Box
- Start Point
2. Transcription unit – sequence that actually is copied into RNA
3. Terminator region

11. Transcription is 3 steps
1. Initiation
2. Elongation
3. Termination

12. 1. INITIATION
Process:
1. Protein transcription factors bind to the TATA box in the promoter region – allows RNA polymerase to bind
2. RNA polymerase binds to the start point
3. Additional transcription factors bind and help RNA polymerase to unwind the DNA
- Transcription factors (proteins) must attach to the TATA box first -

13. Polymerase, Transcription Factors and Promoter together are called the TRANSCRIPTION INITIATION PROCESS

14. Polymerase, Transcription Factors and Promoter together are called the TRANSCRIPTION INITIATION PROCESS

15. Transcription initiation complex
RNA PROCESSING
TRANSLATION
DNA
Pre-mRNA
mRNA
Ribosome
Polypeptide T A
TATA box
Start point
Template
DNA strand 5 3
Transcription
factors
Promoter
RNA polymerase II
Transcription factors
RNA transcript
Transcription initiation complex
Eukaryotic promoters 1
Several transcription 2
Additional transcription 3

16. The attachment of the transcription factors is guided by enhancer sequences of DNA upstream from the transcription unit. Activator proteins bind to the enhancer regions causing the DNA to bend. This guides the transcription factors in place to aid RNA polymerase binding

18. 2. ELONGATION
- building of RNA along DNA Template strand (side of DNA copied is determined by promoter)
- RNA is built in the 5’ to 3’ direction by adding to the 3’ end of the RNA
- RNA polymerase moves along the DNA and builds the RNA COMPLIMENTARY to the DNA sequence (U for T)

20. Transcribe:
GCCATCTAAATCGGG
CGGUAGAUUUAGCCC
- the DNA closes together after the polymerase has passed until the RNA polymerase reaches the termination point

21. 3. TERMINATION
RNA polymerase reaches the terminator segment of DNA and signals for the release of the polymerase from the DNA
- in prokaryotes, this ends transcription
- in eurkaryotes, the polymerase moves past the terminator point slightly onto an AUAAAsequence and then is released - this allows for the modification of the RNA later

22. Figure 17.7 Completed RNA transcript Promoter Transcription unit
RNA polymerase
Start point 5 3
Rewound
RNA
transcript
Completed RNA transcript
Unwound
DNA
Template strand of DNA

23. Transcription Video

24. Basic types of RNA 1. Messenger RNA = mRNA
2. Transfer RNA = tRNA (translation)
3. Ribosomal RNA = rRNA (makes ribosomes)
4. Small nuclear RNA = snRNA (for RNA splicing)
5. Signal Recognition Particle RNA = SRP RNA
– makes ribosomes bind to rough ER

26. 6. Small nucleolar RNA = sno RNA (aid in ribosome formation)
7. Small interfering RNA = siRNA (gene regulation)
8. MicroRNA = miRNA (gene regulation)

28. RNA PROCESSING
Pre-mRNA must undergo modifications before it can leave the nucleus
1. Modification of the ends
2. Removal of Introns and Splicing of Exons

29. 1: modification of the ends
5’ end - promoter end
- capped with a guanine triphosphate (GTP) molecule
Two Functions
1. prevents mRNA from degrading
2. attachment site for Translation
3’ end - terminator end - capped with a Poly A tail
- long chain of adenines
- prevents degradation

30. A modified guanine nucleotide added to the 5 end
50 to 250 adenine nucleotides
added to the 3 end
Protein-coding segment
Polyadenylation signal
Poly-A tail
3 UTR
Stop codon
Start codon
5 Cap
5 UTR
AAUAAA
AAA…AAA
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide G P 5 3

31. 2: Splicing
Eukaryotic DNA is filled with large sections of DNA that are not coded for proteins
- copied in transcription
- must be removed or it would change the resulting polypeptide sequence  shape  function

32. Non-coded DNA = intron (“in” for intervening or interrupting)
- once thought to be “junk DNA” but now thought to have regulatory functions in gene expression
Coded DNA = exon (“ex” for expressed)
- only 3% of the human genome codes for proteins – rest is not expressed (introns), codes for the different RNAs or ????

33. exons spliced together Coding segment
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
5 Cap
Exon
Intron 1 5 30 31
104
105
146 3
Poly-A tail
Introns cut out and
exons spliced together
Coding
segment
3 UTR

34. - pre-mRNA must have the introns removed and splice (join) the exons
Completed By: small nuclear ribonucleoproteins (snRNP  “snurps”)
- snurps - attach to signals at the ends of the introns and, with proteins, form a SPLICEOSOME
- cuts out the intron and attaches the ends of the exons

35. RNA transcript (pre-mRNA)
Exon 1
Intron
Exon 2
Other proteins
Protein
snRNA
snRNPs
Spliceosome
components
Cut-out
intron
mRNA 5 1 2 3
Analogy: pre-mRNA: Watching TV with commercials mRNA: Watching TV on DVD

36. RNA Processing Video

37. TRANSLATION Basics: 1. m-RNA binds to ribosome
2. t-RNA matches up to m-RNA and brings in AA
3. AA chain built
4. polypeptide detaches

38. Amino acids tRNA with amino acid attached Trp Phe Gly tRNA Anticodon
TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
Amino
acids
tRNA with
amino acid
attached
tRNA
Anticodon
Trp
Phe
Gly A G C U
Codons 5 3

39. Background STRUCTURE OF t-RNA:
sequence of RNA transcribed from the DNA - 80 nucleotides long
- specific three dimensional conformation (foldings and turnings) - "L" shape

41. 3' end to attach to amino acids
- series of three nucleotides that match to m-RNA codon
- called ANTI-CODON (complimentary to m-RNA)

43. - matches specifically to the first two nucleotides but the third is not as fixed  WOBBLE effect - can match to one or more bases and therefore GGU and GGC can code for the same amino acid
TOTAL: about 45 different t-RNAs
- bind specifically to one type of amino acid
accomplished by : AMINOACYL-tRNA SYNTHETASE

45. STRUCTURE OF RIBOSOME - rRNA and proteins
2 subunits: large and small - join together when bound to mRNA
Large subunit: 3 binding sites for the building the AA chain
A site - aminoacyl tRNA site
P site - peptidyl tRNA site
E site - exit site

47. Eukaryotic and Prokaryotic Ribosome differences
- eu bigger and slightly different molecularly
allows for targeting by antibiotics

48. TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
Exit tunnel
Growing
polypeptide
tRNA
molecules E P A
Large
subunit
Small
Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins.
(a) 5 3

49. TRANSLATION: the details
3 steps:
1. initiation
2. elongation
3. termination

50. Initiation: Formation of Translation Initiation Complex
1. m-RNA binds to small subunit
on "attach here" signal in 5'cap
2. binding of first amino acid = START METHIONINE
ALWAYS: AUG codon
3. attachment of large subunit
start methionine-tRNA in "P site"

51. process assisted by protein ‘initiation factors’ CODON (m-RNA) triplets are lined up in the different sites of the ribosome - ready for elongation

52. Elongation
1. Codon recognition: next matching tRNA comes in with appropriate AA to A site

54. 2.Peptide bond formation: ribosome transfers AA chain from P site tRNA to A site tRNA - forms a peptide bond by dehydration synthesis powered by GTP

55. 3. Translocation: shifting of ribosome complex down mRNA - tRNAs bonded to mRNA and slide down into next site on ribosome
A to P
P to E
- empty tRNA in E site leaves (exits) leaving the A site open and P site has tRNA with elongated AA chain

56. process continues until Termination

57. Termination Ribosome reaches a STOP codon: UAA, UAG, UGA
- brings a protein called a release factor instead of an amino acid
- binds into A site -
- no transfer of AA acid chain
- hydrolyzes AA chain from tRNA
- tRNA exits
- Ribosome complex disassembles

59. Translation Video
Translation II

60. Polyribosomes:
once initiator complex is open another ribosome can attach

61. FINISHED PRODUCTS
1. Cytosolic ribosomes: Finished proteins take on three dimensional shape with the help of chaperone proteins - direction conformation of protein (post-translational modifications)

62. 2. Bound ribosomes:
- start as cytosolic ribosomes
- at beginning of translation, the mRNA codes for a signal peptide that attaches to SIGNAL RECOGNITION PARTICLE (SRP)
- brings ribosome into the ER
- Growing polypeptide chain enters the ER cisternal space
- secretion proteins enter the whole way
- membrane proteins stay bound to the ER membrane

64. CHANGES IN THE DNA = mutation
Chromosomal mutations: changes that affect the whole chromosome
Point mutation: change that affects only one nucleotide in the DNA
EXAMPLE:
Hemoglobin and Sickle cell Hemoglobin
In normal DNA code is CTT which is made into RNA (GAA)
In Sickle cell the DNA code is CAT - second T is replaced with A - therefore RNA becomes GUA
GAA = glutamine GUA = valine
- one nucleotide changes one amino acid which changes the shape of the protein which changes the shape of the cell which causes all kinds of health defects

65. the wild-type template has a T.
In the DNA, the
mutant template
strand has an A where
the wild-type template
has a T.
The mutant mRNA has
a U instead of an A in
one codon.
The mutant (sickle-cell)
hemoglobin has a valine
(Val) instead of a glutamic
acid (Glu).
Mutant hemoglobin DNA
Wild-type hemoglobin DNA
mRNA
Normal hemoglobin
Sickle-cell hemoglobin
Glu
Val C T A G U 3 5

66. TYPES OF POINT MUTATIONS
Substitution: one base pair replaces another
1. Silent - WOBBLE effect
Ex: CCG changed to CCA
Result: GGC changed to GGU
- both bring in glycine

67. Base-pair substitution
Wild type A U G C
mRNA 5
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end 3
Base-pair substitution
No effect on amino acid sequence
U instead of C
Ser
Missense
A instead of G
Nonsense
U instead of A

68. 2. Missense: replacement of base pair that brings in another amino acid that changes the structure of the protein
Ex: Sickle cell

69. Base-pair substitution
Wild type A U G C
mRNA 5
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end 3
Base-pair substitution
No effect on amino acid sequence
U instead of C
Ser
Missense
A instead of G
Nonsense
U instead of A

70. 3. Non-sense: substitution where change in DNA sequence brings in a stop codon in the middle of the protein - protein never is finished

71. Base-pair substitution
Wild type A U G C
mRNA 5
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end 3
Base-pair substitution
No effect on amino acid sequence
U instead of C
Ser
Missense
A instead of G
Nonsense
U instead of A

72. Frameshift Mutations
1. Insertion - additional nucleotide is added to DNA shifting the amino acid sequence - changes all amino acids after insertion

74. 2. Deletion - same affect due to loss of nucleotide
Frameshifts (both insertion and deletion) are usually most devastating if its one or two nucleotides
- insertion or deletion of a triplet may only result in the loss or gain of one amino acid - may or may not disrupt protein