Gen Bio II BIO102

1. Chapter 17
From Gene to Protein
Rob Swatski
Associate Professor of Biology
HACC – York Campus

2. Overview: The Flow of Genetic
Information
DNA information = specific sequences of nucleotides
DNA  protein synthesis
Proteins: link genotype & phenotype
Gene expression: DNA directs protein synthesis
- 2 stages: transcription & translation

3. How was the fundamental relationship between
genes & proteins discovered?
- examine evidence from studies of
metabolic defects

4. 1909: British physician Archibald
Garrod 1st suggested that genes
dictate phenotypes with
enzymes
- symptoms of inherited disease
reflects inability to synthesize a
certain enzyme
- required understanding that cells
synthesize & degrade molecules
using metabolic pathways

5. Neurospora
bread mold

6. Nutritional Mutants in Bread Mold
George Beadle & Edward Tatum exposed Neurospora
to x-rays
- created mutants that could not survive on minimal
medium
(cannot synthesize certain molecules)
Identified 3 classes of arginine-deficient mutants
- each lacked a different enzyme needed to make
arginine
Developed the “one gene – one enzyme hypothesis”
- each gene directs the synthesis of a specific enzyme

7. Minimal medium
No growth:
Mutant cells
cannot grow
and divide
Growth:
Wild-type
cells growing
and dividing
EXPERIMENT

8. RESULTS
Classes of Neurospora crassa
Wild type Class I mutants Class II mutants Class III mutants
Minimal
medium
(MM)
(control)
MM 
ornithine
MM 
citrulline
Condition
MM 
arginine
(control)
Summary
of results
Can grow with
or without any
supplements
Can grow on
ornithine,
citrulline, or
arginine
Can grow only
on citrulline or
arginine
Require arginine
to grow
Growth
No
growth

9. CONCLUSION
Wild type
Class I mutants
(mutation in
gene A)
Class II mutants
(mutation in
gene B)
Class III mutants
(mutation in
gene C)
Gene
(codes for
enzyme)
Gene A
Gene B
Gene C
Precursor Precursor Precursor Precursor
Enzyme A Enzyme A Enzyme A Enzyme A
Enzyme B Enzyme B Enzyme B Enzyme B
Ornithine Ornithine Ornithine Ornithine
Enzyme C Enzyme C Enzyme CEnzyme C
Citrulline Citrulline Citrulline Citrulline
Arginine Arginine Arginine Arginine

10. The Products of Gene Expression:
A Developing Story
• Some proteins aren’t enzymes… so researchers
later revised the hypothesis to “one gene – one
protein”
• But, many proteins consist of several polypeptides,
each having its own gene
Beadle & Tatum’s hypothesis is now restated as the:
“one gene–one polypeptide hypothesis”

11. Basics of Transcription & Translation
RNA: the bridge between genes & the proteins they
code
• Transcription: synthesis of RNA under the direction
of DNA
- produces messenger RNA (mRNA)
• Translation: synthesis of a polypeptide under the
direction of mRNA
- ribosomes: the sites of translation

12. Eukaryotes:
the nuclear envelope separates transcription from
translation
- eukaryotic RNA transcripts are modified via RNA
processing to yield finished mRNA
Prokaryotes:
mRNA transcripts are immediately translated without
further processing

13. (a) Prokaryotic Cell (Bacteria)
TRANSCRIPTION
DNA
mRNA
TRANSLATION
Ribosome
Polypeptide

14. Primary transcript: initial RNA transcript from a gene
before processing
Central dogma:
cells are governed by a cellular chain of command:
DNA RNA Protein

15. (b) Eukaryotic cell
TRANSCRIPTION
Nuclear
envelope
DNA
Pre-mRNA
RNA PROCESSING
mRNA

16. (b) Eukaryotic cell
TRANSCRIPTION
Nuclear
envelope
DNA
Pre-mRNA
RNA PROCESSING
mRNA
TRANSLATION Ribosome
Polypeptide

17. The Genetic Code
How are the instructions for assembling amino acids
into proteins encoded into DNA?
There are 20 amino acids,
… but there are only 4 nucleotide bases in DNA
How many bases correspond to an amino acid?

18. Codons: Triplets of Nucleotides
The flow of information from gene  protein is based
on a triplet code
- series of non-overlapping 3-nucleotide “words”
Triplet: smallest unit that can code for amino acids
- “AGT” = placement of serine at its correct position in
the polypeptide

19. Transcription:
one of the two DNA strands (template strand)
provides a pattern for ordering the nucleotide
sequence in the mRNA transcript
Translation:
mRNA base triplets (codons) are read in the 5 to 3
direction
- each codon specifies the amino acid (1 of 20) and
it’s correct position in a polypeptide

20. DNA
template
strand
TRANSCRIPTION
mRNA
TRANSLATION
Protein
Amino acid
Codon
Trp Phe Gly
5
5
Ser
U U U U U
3
3
53
G
G
G G C C
T
C
A
A
AAAAA
T T T T
T
G
G G G
C C C G G
DNA
molecule
Gene 1
Gene 2
Gene 3
C C

21. Cracking the Code
• All 64 codons were deciphered by the mid-1960s
• Of the 64 triplets, 61 code for amino acids
- 3 triplets are stop codons that end translation:
UAA, UAG, UGA
• The genetic code is redundant but not ambiguous
• Codons must be read in the correct reading frame
(correct groupings) in order to synthesize the
specified polypeptide

22. Second mRNA base
FirstmRNAbase(5endofcodon)
ThirdmRNAbase(3endofcodon)
UUU
UUC
UUA
CUU
CUC
CUA
CUG
Phe
Leu
Leu
Ile
UCU
UCC
UCA
UCG
Ser
CCU
CCC
CCA
CCG
UAU
UAC
Tyr
Pro
Thr
UAA Stop
UAG Stop
UGA Stop
UGU
UGC
Cys
UGG Trp
GC
U
U
C
A
U
U
C
C
C
A
U
A
A
A
G
G
His
Gln
Asn
Lys
Asp
CAU CGU
CAC
CAA
CAG
CGC
CGA
CGG
G
AUU
AUC
AUA
ACU
ACC
ACA
AAU
AAC
AAA
AGU
AGC
AGA
Arg
Ser
Arg
Gly
ACGAUG AAG AGG
GUU
GUC
GUA
GUG
GCU
GCC
GCA
GCG
GAU
GAC
GAA
GAG
Val Ala
GGU
GGC
GGA
GGG
Glu
Gly
G
U
C
A
Met or
start
UUG
G

23. Evolution of the Genetic Code
The genetic code is shared by all living things
- Genes can be transcribed & translated after
being transplanted from one species to another

24. (a) Tobacco plant expressing a firefly gene

25. (b) Pig expressing a jellyfish gene

26. Synthesis of an RNA Transcript
The 3 Stages of Transcription:
1. Initiation
2. Elongation
3. Termination

27. Transcription: DNA  RNA
RNA synthesis is catalyzed by RNA polymerase
- pries DNA strands apart
- hooks RNA nucleotides together
The RNA is complementary to the DNA template
strand
Follows same base-pairing rules as DNA
- except uracil substitutes for thymine

28. Promoter: DNA sequence that RNA polymerase
attaches to
Transcription unit: section of DNA that is
transcribed

29. Promoter Transcription unit
DNA
Start point
RNA polymerase
5
5
3
3
Initiation
3
3
1
RNA
transcript
5
5
Unwound
DNA
Template strand
of DNA
2
Elongation
Rewound
DNA
5
5
53 3 3
RNA
transcript
3
Termination
5
5
53
3
3
Completed RNA transcript

30. RNA Polymerase Binding & Initiation
of Transcription
Promoters: signal initiation of RNA synthesis
- TATA box promoter is crucial in forming the initiation
complex in eukaryotes
Transcription factors:
needed to help bind RNA polymerase & initiate
transcription
Transcription initiation complex:
completed assembly of transcription factors & RNA
polymerase bound to a promoter

31. Transcription initiation complex forms3
DNA
Promoter
Nontemplate strand
5
3
5
3
5
3
Transcription
factors
RNA polymerase II
Transcription factors
5
3
5
3
5
3
RNA transcript
Transcription initiation complex
5
3
TATA box
T
T T T T T
A A A A A
A A
T
Several transcription factors bind to DNA2
A eukaryotic promoter1
Start point Template strand

32. Elongation of the RNA Strand
As RNA polymerase moves along DNA, it untwists the
double helix, 10-20 bases at a time
- transcription rate = 40 nucleotides/sec
A gene can be transcribed simultaneously by several
RNA polymerases
Nucleotides are added to the 3’ end of the growing
RNA molecule

33. Elongation
RNA
polymerase
Non-template
strand of DNA
RNA nucleotides
3 end
Direction of
transcription
(“downstream”) Template
strand of DNA
Newly made
RNA
3
5
5

34. Termination of Transcription
In bacteria:
RNA polymerase stops transcription at end of the
terminator & the mRNA can be translated
without further modification
In eukaryotes:
RNA polymerase continues transcription after the
pre-mRNA is cleaved from the growing RNA
chain
- polymerase eventually falls off the DNA

35. Eukaryotic Cells Modify RNA
After Transcription
Enzymes in the eukaryotic nucleus modify pre-mRNA
(RNA processing) before mRNA “gene” enters
cytoplasm
Both ends of the primary transcript are usually altered
- and some interior parts of RNA are usually cut-out &
other parts spliced together

36. Alteration of mRNA Ends
Each end of pre-mRNA is modified in a particular way:
- the 5 end gets a modified nucleotide 5 cap
- the 3 end gets a poly-A tail
Why Modify?
- Facilitates export of mRNA
- Protects mRNA from hydrolytic enzymes
- Helps ribosomes attach to the 5 end

37. Protein-coding
segment
Polyadenylation
signal
5 3
35 5Cap UTR Start
codon
G P P P
Stop
codon
UTR
AAUAAA
Poly-A tail
AAA AAA…

38. Split Genes & RNA Splicing
Most genes & their RNA transcripts have long
noncoding regions (introns) that lie between
coding regions
- intron = intervening sequences (“in the way”)
Exons: coding regions
- expressed & translated into amino acid sequences
RNA splicing: removes introns & joins exons
- creates mRNA molecule with a continuous
coding sequence

39. 5 Exon Intron Exon
5 CapPre-mRNA
Codon
numbers
130 31104
mRNA 5 Cap
5
Intron Exon
3 UTR
Introns cut out and
exons spliced together
3
105
146
Poly-A tail
Coding
segment
Poly-A tail
UTR
1146

40. Some RNA splicing is carried out by
spliceosomes
- consist of a variety of proteins & small nuclear
ribonucleoproteins (snRNPs = “snurps”)
- snRNPs can recognize the splice sites

41. RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
Protein
snRNA
snRNPs
Other
proteins
5
5
Spliceosome
Spliceosome
components
Cut-out
intron
mRNA
Exon 1 Exon 2
5

42. Ribozymes
Catalytic RNA molecules that act as enzymes & splice RNA
- not all biological catalysts are proteins!

43. How can RNA function as an enzyme?
- can form a 3-D structure because it can base-pair with
itself
- some RNA bases contain functional groups
- can hydrogen-bond with other nucleic acids

44. Some genes can encode more than 1 kind of
polypeptide, depending on which segments are
treated as exons during RNA splicing
- the actual # of different proteins an organism can
produce is much greater than its number of genes
Alternative RNA Splicing

45. Proteins often have a modular architecture consisting
of discrete regions called domains
- different exons can code for different domains in a
protein
Exon shuffling can result in the evolution of new
proteins

46. Gene
DNA
Exon 1 Exon 2 Exon 3Intron Intron
Transcription
RNA processing
Translation
Domain 2
Domain 3
Domain 1
Polypeptide

47. Molecular Components of Translation
A cell translates mRNA message into protein with
the help of transfer RNA (tRNA)
tRNA molecules are not identical:
- each carries a specific amino acid on one end
- each has an anticodon on the other end that base-
pairs with a complementary codon on mRNA

48. Polypeptide
Ribosome
Amino
acids
tRNA with
amino acid
attached
tRNA
Anticodon
Codons 35
mRNA

49. The Structure & Function of tRNA
tRNA: one RNA
strand, 80 nucleotides long
- when flattened, it resembles
a cloverleaf
C
Amino acid
attachment site
Hydrogen
bonds
Anticodon
3
5

50. Can twist & fold into an “L”-shaped 3-D molecule
through hydrogen-bonding
Amino acid
attachment site
3
3
5
5
Hydrogen
bonds
Anticodon Anticodon
(b) 3-D structure
(c) Symbol used
in this book

51. Translation requires 2 steps: “The Match Game”
1. tRNA and its amino acid are matched by the enzyme
aminoacyl-tRNA synthetase
- forms “charged tRNA”
2. tRNA anticodon and an mRNA codon are matched
Flexible pairing at the 3rd base of a codon is called wobble
- allows some tRNAs to bind to more than 1 codon

52. Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P Adenosine
ATP
P
P
P
P
Pi
i
i
Adenosine
tRNA
AdenosineP
tRNA
AMP
Computer model
Amino
acid
Aminoacyl-tRNA
synthetase
Aminoacyl tRNA
(“charged tRNA”)

53. Ribosomes
Ribosomes facilitate specific coupling of tRNA
anticodons with mRNA codons in protein synthesis
- 2 ribosomal subunits (large & small) are made of
proteins & ribosomal RNA (rRNA)

54. Growing
polypeptide
Exit tunnel
tRNA
molecules
Large
subunit
Small
subunit
(a) Computer model of functioning ribosome
mRNA
E P A
5
3

56. Exit tunnel
A site (Aminoacyl-
tRNA binding site)
Small
subunit
Large
subunit
P A
P site (Peptidyl-tRNA
binding site)
mRNA
binding site
(b) Schematic model showing binding sites
E site
(Exit site)
E

57. A ribosome has 3 binding sites for tRNA:
- A site: holds the tRNA carrying the next amino acid to be
added to the chain
- P site: holds the tRNA carrying the growing polypeptide
chain
- E site (Exit): where discharged tRNAs leave the ribosome

58. Amino end
mRNA
E
(c) Schematic model with mRNA and tRNA
5 Codons
3
tRNA
Growing polypeptide
Next amino
acid to be
added to
polypeptide
chain

59. Building a Polypeptide
The 3 stages of translation:
1. Initiation
2. Elongation
3. Termination
All 3 stages require protein factors

60. Initiation of Translation
Initiation stage: brings together mRNA, a tRNA with
the 1st amino acid, & the 2 ribosomal subunits
1. First, the small ribosomal subunit binds with mRNA
and a special initiator tRNA
2. Then the small subunit moves along mRNA until it
reaches the start codon (AUG)
- Initiation factors bring in the large subunit to
complete the translation initiation complex

61. 3
35
5U
U
A
A
C
G
GTP GDP
Initiator
tRNA
mRNA
5 3
Start codon
mRNA binding site
Small
ribosomal
subunit
5
P site
Translation initiation complex
3
E A
Large
ribosomal
subunit

62. Elongation of the Polypeptide Chain
Elongation stage: amino acids are added one by one
Each addition involves elongation factors and occurs
in 3 steps:
a. Codon recognition
b. Peptide bond formation
c. Translocation

63. Amino end
of polypeptide
mRNA
5
3E
P
site
A
site
GTP
GDP
E
P A

64. Amino end
of polypeptide
mRNA
5
3E
P
site
A
site
GTP
GDP
E
P A
E
P A

65. Amino end
of polypeptide
mRNA
5
3E
P
site
A
site
GTP
GDP
E
P A
E
P A
GDP
GTP
Ribosome ready for
next aminoacyl tRNA
E
P A

66. Termination of Translation
Termination:
- occurs when a stop codon in mRNA reaches the A site
The A site accepts a release factor protein
- adds a water molecule instead of an amino acid
- this releases the polypeptide
- the translation complex comes apart

67. Release
factor
3
5
Stop codon
(UAG, UAA, or UGA)
5
3
2
Free
polypeptide
2 GDP
GTP
5
3

68. Polyribosomes
Groups of ribosomes that simultaneously translate one
mRNA, forming a polyribosome (polysome)
- allows a cell to quickly make many copies of a
polypeptide

69. Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
End of
mRNA
(3 end)
(a)
Ribosomes
mRNA
(b) 0.1 µm

70. Post-Translation
A protein is usually not functional immediately
after translation
- requires further post-translational
modification
It spontaneously coils and folds into its correct
3-D shape
- some activated by enzymes that cleave them
- others assemble into protein subunits

71. Targeting Polypeptides to Specific
Locations
Two populations of ribosomes are found in cells:
- Free ribosomes: synthesize proteins that function in
the cytosol
- Bound ribosomes: synthesize proteins on ER and
those that will be secreted from the cell
Ribosomes are identical and can switch from free to
bound

72. Polypeptide synthesis always begins and ends in
the cytosol
- unless the polypeptide signals the ribosome to
attach to the ER
Polypeptides destined for the ER or for secretion
are marked by a signal peptide
- a signal-recognition particle (SRP) binds to the
signal peptide
- the SRP brings the signal peptide & its
ribosome to the ER

73. Ribosome
mRNA
Signal
peptide
Signal-
recognition
particle (SRP)
CYTOSOL
Translocation
complex
SRP
receptor
protein
ER LUMEN
Signal
peptide
removed
ER
membrane
Protein

74. Point Mutations
- chemical changes in just 1 base pair of a gene
- a change in one DNA nucleotide can lead to the production of
an abnormal protein
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
3
3
3
3
3
3
5
5
5
5
5
5
C CT T T
TG GA A A
A
A A AGG U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val

75. Types of Point Mutations
- Base-pair substitutions
- Base-pair insertions or deletions

76. Wild type
3DNA template
strand
3
35
5
5mRNA
Protein
Amino end
Stop
Carboxyl end
A instead of G
3
3
3
U instead of C
5
5
5
Stop
Silent mutations: have no effect on the amino acid
because of redundancy in the genetic code

77. Wild type
DNA template
strand
3
5
mRNA
Protein
5
Amino end
Stop
Carboxyl end
5
3
3
T instead of C
A instead of G
3
3
3
5
5
5
Stop
Missense: still codes for an amino acid, but not
necessarily the right amino acid

78. Wild type
DNA template
strand
3
5
mRNA
Protein
5
Amino end
Stop
Carboxyl end
5
3
3
A instead of T
U instead of A
3
3
3
5
5
5
Stop
Nonsense: changes an amino acid codon into a stop codon,
nearly always leading to a nonfunctional protein

79. Insertions and Deletions
- additions or losses of nucleotide pairs in a gene
Can have a disastrous effect on the protein more often
than substitutions
- may produce a frameshift mutation, which alters the
reading frame

80. Wild type
DNA template
strand
3
5
mRNA
Protein
5
Amino end
Stop
Carboxyl end
5
3
3
Extra A
Extra U
3
3
3
5
5
5
Stop
Frameshift Mutation causing immediate nonsense
(1 base-pair insertion)

81. Wild type
DNA template
strand
3
5
mRNA
Protein
5
Amino end
Stop
Carboxyl end
5
3
3
missing
missing
3
3
3
5
5
5
Frameshift Mutation causing extensive missense
(1 base-pair deletion)

82. Wild type
DNA template
strand
3
5
mRNA
Protein
5
Amino end
Stop
Carboxyl end
5
3
3
missing
missing
3
3
3
5
5
5
No Frameshift Mutation, but one amino acid missing
(3 base-pair deletion)
Stop

83. What Is a Gene?
We have considered a gene as:
- A discrete unit of inheritance
- A region of specific nucleotide sequence in a
chromosome
- A DNA sequence that codes for a specific polypeptide
chain
In summary, a gene can be defined as:
- a region of DNA that can be expressed to
produce a final functional product, either a
polypeptide or an RNA molecule

84. TRANSCRIPTION
RNA PROCESSING
DNA
RNA
transcript
3
5
RNA
polymerase
RNA transcript
(pre-mRNA)
Intron
Exon
NUCLEUS
Aminoacyl-tRNA
synthetase
AMINO ACID ACTIVATION
Amino
acid
tRNACYTOPLASM
Growing
polypeptide
3
Activated
amino acid
mRNA
TRANSLATION
Ribosomal
subunits
5
E
P
A
A
Anticodon
Ribosome
Codon
E