1. From Gene to Protein How Genes Work

2. Making proteins Organelles nucleus ribosomes
endoplasmic reticulum (ER)
Golgi apparatus
vesicles
small
ribosomal
subunit
nuclear pore
mRNA
large
ribosomal
subunit
cytoplasm

3. Nucleolus Function ribosome production
build ribosome subunits from rRNA & proteins
exit through nuclear pores to cytoplasm & combine to form functional ribosomes
small
subunit
large subunit
ribosome
rRNA &
proteins
nucleolus

4. Ribosomes Function Structure protein production rRNA & protein
small
subunit
large
Ribosomes
Function
protein production
Structure
rRNA & protein
2 subunits combine
0.08mm
Ribosomes
Rough ER
Smooth
The genes for rRNA have the greatest commonality among all living things. There is very little difference in the DNA sequence of the rRNA genes in a humans vs. a bacteria.
Means that this function (building of a ribosome) is so integral to life that every cell does it almost exactly the same way.
Change a base and this changes the structure of the RNA which causes it to not function.
rRNA genes are always turned on.

5. Types of Ribosomes Free ribosomes Bound ribosomes suspended in cytosol
synthesize proteins that function in cytosol
Bound ribosomes
attached to endoplasmic reticulum
synthesize proteins for export or for membranes
membrane proteins

6. endoplasmic reticulum
TO:
nucleus
protein on its way!
TO:
DNA
RNA
vesicle
TO:
TO:
vesicle
ribosomes
TO:
protein
finished protein
Golgi apparatus
Making Proteins

7. Metabolism taught us about genes
Inheritance of metabolic diseases
suggested that genes coded for enzymes
each disease (phenotype) is caused by non-functional gene product
lack of an enzyme
Tay sachs
PKU (phenylketonuria)
albinism
Am I just the sum of my proteins?
metabolic pathway
disease
disease
disease
disease A B C D E    
enzyme 1
enzyme 2
enzyme 3
enzyme 4

8. one gene : one enzyme hypothesis
1941 | 1958
Beadle & Tatum
one gene : one enzyme hypothesis
George Beadle
Edward Tatum
"for their discovery that genes act by regulating definite chemical events"

9. DNA gets all the glory, but proteins do all the work!
The “Central Dogma”
Flow of genetic information in a cell
How do we move information from DNA to proteins?
transcription
translation
DNA
RNA
protein
trait
To get from the chemical language of DNA to the chemical language of proteins requires 2 major stages:
transcription and translation
DNA gets all the glory, but proteins do all the work!
replication

10. DNA RNA RNA ribose sugar N-bases single stranded lots of RNAs
uracil instead of thymine
U : A
C : G
single stranded
lots of RNAs
mRNA, tRNA, rRNA, siRNA…
transcription
DNA
RNA

11. from DNA nucleic acid language to RNA nucleic acid language
Transcription
from DNA nucleic acid language to RNA nucleic acid language

12. Transcription Making mRNA transcribed DNA strand = template strand
untranscribed DNA strand = coding strand
same sequence as RNA
synthesis of complementary RNA strand
transcription bubble
enzyme
RNA polymerase
coding strand 3 A G C A T C G T 5 A G A A A G T C T T C T C A T A C G
DNA T 3 C G T A A T 5 G G C A U C G U T 3 C
unwinding G T A G C A
rewinding
mRNA
RNA polymerase
template strand
build RNA 53 5

13. Transcription in Eukaryotes
RNA Processing
Psssst… DNA can’t leave nucleus!
Translation
Protein

14. Prokaryote vs. Eukaryote genes
Prokaryotes
DNA in cytoplasm
circular chromosome
naked DNA
no introns
Eukaryotes
DNA in nucleus
linear chromosomes
DNA wound on histone proteins
introns vs. exons
Walter Gilbert hypothesis:
Maybe exons are functional units and introns make it easier for them to recombine, so as to produce new proteins with new properties through new combinations of domains.
Introns give a large area for cutting genes and joining together the pieces without damaging the coding region of the gene…. patching genes together does not have to be so precise.
introns come out!
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence

15. Transcription in Eukaryotes
3 RNA polymerase enzymes
RNA polymerase 1
only transcribes rRNA genes
makes ribosomes
RNA polymerase 2
transcribes genes into mRNA
RNA polymerase 3
only transcribes tRNA genes
each has a specific promoter sequence it recognizes

16. Transcription in Eukaryotes
Initiation complex
transcription factors bind to promoter region upstream of gene
suite of proteins which bind to DNA
turn on or off transcription
TATA box binding site
recognition site for transcription factors
transcription factors trigger the binding of RNA polymerase to DNA

17. Post-transcriptional processing
Primary transcript (pre-mRNA)
eukaryotic mRNA needs work after transcription
mRNA processing (making mature mRNA)
mRNA splicing = edit out introns
protect mRNA from enzymes in cytoplasm
add 5 cap
add polyA tail
3' poly-A tail 3' A A A A A
5' cap
mRNA
A’s P P P 5' G
intron = noncoding (inbetween) sequence
eukaryotic RNA is about 10% of eukaryotic gene.
~10,000 bases
eukaryotic DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
~1,000 bases
mature mRNA
transcript
spliced mRNA

18. Splicing must be accurate
No room for mistakes!
splicing must be exactly accurate
a single base added or lost throws off the reading frame
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|UCC|GAU|AAG|GGC|CAU
Met|Arg|Ser|Asp|Lys|Gly|His
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
Met|Arg|Val|Arg|STOP|

19. we just broke a biological “rule”!
Whoa! I think
we just broke a biological “rule”!
Splicing enzymes
snRNPs
small nuclear RNA
proteins
Spliceosome
several snRNPs
recognize splice site sequence
cut & paste
snRNPs
exon
intron
snRNA 5' 3'
spliceosome
exon
excised
intron 5' 3'
lariat
mature mRNA

20. Ribozyme 1982 | 1989 RNA as ribozyme some mRNA can even splice itself
RNA as enzyme
Sidney Altman
Thomas Cech
Yale
U of Colorado

21. from nucleic acid language to amino acid language
Translation
from nucleic acid language to amino acid language

22. Translation
Codons
blocks of 3 nucleotides decoded into the sequence of amino acids

23. Translation: prokaryotes vs. eukaryotes
Differences between prokaryotes & eukaryotes
time & physical separation between processes
takes eukaryote ~1 hour from DNA to protein
RNA processing

24. Translation in Eukaryotes

25. DNA mRNA protein From gene to protein transcription translationaa
transcription
translation
DNA
mRNA
protein
mRNA leaves nucleus through nuclear pores
ribosome
proteins synthesized by ribosomes using instructions on mRNA
nucleus
cytoplasm

26. How does mRNA code for proteins?
TACGCACATTTACGTACGCGG
DNA 4
ATCG
AUGCGUGUAAAUGCAUGCGCC
mRNA 4
AUCG ?
Met Arg Val Asn Ala Cys Ala
protein 20
How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

27. mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGG
DNA
codon
AUGCGUGUAAAUGCAUGCGCC
mRNA
AUGCGUGUAAAUGCAUGCGCC
mRNA ?
Met Arg Val Asn Ala Cys Ala
protein

28. WHYDIDTHEREDBATEATTHEFATRAT WHYDIDTHEREDBATEATTHEFATRAT
1960 | 1968
Cracking the code
Nirenberg & Khorana
Crick
determined 3-letter (triplet) codon system
WHYDIDTHEREDBATEATTHEFATRAT
WHYDIDTHEREDBATEATTHEFATRAT
Nirenberg (47) & Khorana (17)
determined mRNA–amino acid match
added fabricated mRNA to test tube of ribosomes, tRNA & amino acids
created artificial UUUUU… mRNA
found that UUU coded for phenylalanine (phe)

29. The code Code for ALL life! Code is redundant Start codon Stop codons
strongest support for a common origin for all life
Code is redundant
several codons for each amino acid
3rd base “wobble”
Why is the wobble good?
Strong evidence for a single origin in evolutionary theory.
Start codon
AUG
methionine
Stop codons
UGA, UAA, UAG

30. How are the codons matched to amino acids?3 5
DNA
TACGCACATTTACGTACGCGG 5 3
mRNA
AUGCGUGUAAAUGCAUGCGCC
codon 3 5
tRNA
UAC
Met
GCA
Arg
amino acid
CAU
Val
anti-codon

31. DNA mRNA protein From gene to protein transcription translationaa
transcription
translation
DNA
mRNA
protein
ribosome
nucleus
cytoplasm

32. Transfer RNA structure
“Clover leaf” structure
anticodon on “clover leaf” end
amino acid attached on 3 end

33. tryptophan attached to tRNATrp tRNATrp binds to UGG condon of mRNA
Loading tRNA
Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNA
bond requires energy
ATP  AMP
energy stored in tRNA-amino acid bond
unstable
so it can release amino acid at ribosome easily
The tRNA-amino acid bond is unstable. This makes it easy for the tRNA to later give up the amino acid to a growing polypeptide chain in a ribosome.
Trp
C=O
Trp
Trp
C=O OH
H2O OH O
C=O O
activating
enzyme
tRNATrp A C C U G G
mRNA
anticodon
tryptophan attached to tRNATrp
tRNATrp binds to UGG condon of mRNA

34. Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon
organelle or enzyme?
Structure
ribosomal RNA (rRNA) & proteins
2 subunits
large
small E P A

35. Ribosomes A site (aminoacyl-tRNA site) P site (peptidyl-tRNA site)
holds tRNA carrying next amino acid to be added to chain
P site (peptidyl-tRNA site)
holds tRNA carrying growing polypeptide chain
E site (exit site)
empty tRNA leaves ribosome from exit site
Met U A C 5' U G A 3' E P A

36. Building a polypeptide1 2 3
Building a polypeptide
Initiation
brings together mRNA, ribosome subunits, initiator tRNA
Elongation
adding amino acids based on codon sequence
Termination
end codon
Leu
Val
release
factor
Ser
Met
Met
Met
Met
Leu
Leu
Leu
Ala
Trp
tRNA C A G U A C U A C G A C A C G A C A 5' U 5' U A C G A C 5' A A A U G C U G U A U G C U G A U A U G C U G A A U 5' A A U
mRNA A U G C U G 3' 3' 3' 3' A C C U G G U A A E P A 3'

37. start of a secretory pathway
Destinations:
secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
etc…
Protein targeting
Signal peptide
address label
start of a secretory pathway

38. Can you tell the story? RNA polymerase DNA amino acids tRNA pre-mRNA
exon
intron
tRNA
pre-mRNA
5' cap
mature mRNA
aminoacyl tRNA synthetase
polyA tail 3'
large ribosomal subunit
polypeptide 5'
tRNA
small ribosomal subunit E P A
ribosome

39. Got Questions? Can I translate that for you?

40. Transcription in Prokaryotes
Bacterial chromosome
Transcription in Prokaryotes
Transcription
mRNA
Psssst… no nucleus!
Cell
membrane
Cell wall

41. Transcription in Prokaryotes
Initiation
RNA polymerase binds to promoter sequence on DNA
Role of promoter
Starting point
where to start reading
start of gene
Template strand
which strand to read
Direction on DNA
always read DNA 35
build RNA 53

42. Transcription in Prokaryotes
Promoter sequences
enzyme subunit
RNA polymerase
read DNA 35
bacterial DNA
Promoter
TTGACA
TATAAT
–35 sequence
–10 sequence
RNA polymerase molecules bound to bacterial DNA
RNA polymerase
strong vs. weak promoters

43. Transcription in Prokaryotes
Elongation
RNA polymerase copies DNA as it unwinds
~20 base pairs at a time
bases in gene
builds RNA 53
Simple proofreading
1 error/105 bases
make many mRNAs
mRNA has short life
not worth editing!
reads DNA 35

44. Transcription in Prokaryotes
Termination
RNA polymerase stops at termination sequence
RNA GC hairpin turn

45. Translation in Prokaryotes
Bacterial chromosome
Translation in Prokaryotes
Transcription
mRNA
Translation
Psssst… no nucleus!
protein
Cell
membrane
Cell wall

46. Translation in Prokaryotes
Transcription & translation are simultaneous in bacteria
DNA is in cytoplasm
no mRNA editing
ribosomes read mRNA as it is being transcribed