Ch 17
From Gene to Protein
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The “Central Dogma”
• Flow of genetic information in a cell
– How do we move information from DNA to
proteins?
DNA
RNA
replication
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
protein
DNA gets
all the glory,
but proteins do
all the work!
trait
Making proteins
• Organelles
– nucleus
– ribosomes
– endoplasmic reticulum
(ER)
– Golgi apparatus
– vesicles
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nuclear pore
small
ribosomal
subunit
mRNA
large
ribosomal
subunit
cytoplasm
Nucleolus
• Function
– ribosome production
• build ribosome subunits from rRNA & proteins
• exit through nuclear pores to cytoplasm &
combine to form functional ribosomes
large subunit
small
subunit
rRNA &
proteins
ribosome
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nucleolus
Ribosomes
•
Function- protein production
•
Structure- rRNA & protein; 2 subunits
large
subunit
small
subunit
Free ribosomes
suspended in cytosol
synthesize proteins that function in cytosol
Bound ribosomes
attached to endoplasmic reticulum
synthesize proteins
for export or for membranes
0.08mm
Ribosomes
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Rough
ER
Smooth
ER
Protein Synthesis
• Protein synthesis
- Process of translating amino acid
sequences from a transcription
mRNA unit
• Amino Acid
- Monomer of protein
• Peptide bond
- Links amino acids together
- Created at ribosome during translation
• Polypeptide chain
- Long chain of amino acids
- Once released from ribosome,
it is a protein
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Amino Acids
• Monomer of Protein
• 20 naturally occuring
• Amino and Carboxyl
group, central
carbon with a
hydrogen and a
side chain (R)
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Metabolism taught us about genes
• Inheritance of metabolic diseases
– suggested that genes coded for enzymes
– each disease (phenotype) is caused by nonfunctional gene product
• lack of an enzyme
• Tay sachs
• PKU (phenylketonuria)
• albinism
metabolic pathway
disease
A
disease
disease
disease
B 
C 
D 
E

enzyme 1 enzyme 2 enzyme 3 enzyme 4
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Beadle & Tatum:
one gene – one enzyme hypothesis
Inheritance of metabolic diseases
Some genes coded for enzymes
each disease (phenotype) is
caused by non-functional gene
product
Lack of an enzyme:
PKU (phenylketonuria)
George Beadle
Edward Tatum
"for their discovery that genes act by
regulating definite chemical events"
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
1 gene – 1 enzyme hypothesis
•
Beadle & Tatum
–
Compared mutants of bread mold, Neurospora fungus
• created mutations by X-ray treatments
– X-rays break DNA, damage a gene
• wild type grows on minimal media
– sugars + required nutrients allows fungus to synthesize essential
amino acids
• mutants require added amino acids
– each type of mutant lacks a certain enzyme
needed to
produce a certain amino acid
– non-functional enzyme from damaged gene
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
After Beadle and Tatum …
• Later research refined the one gene - one
enzyme hypothesis.
• First, it became clear that not all proteins are
enzymes and yet their synthesis depends on
specific genes.
Hypothesis modified to one gene - one protein
• Later research demonstrated that many
proteins are composed of several polypeptides,
each of which has its own gene.
• Beadle and Tatum’s idea was restated as the
one gene - one polypeptide hypothesis
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA  RNA  Protein
• Transcription
- one DNA strand used
as template to create
RNA transcript
- Uracil replaces (T)
- Occurs in nucleus
• Translation
- RNA transcript decoded
into a sequence of
amino acids (protein)
- Occurs in cytosol
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Codon and the Decoding Box
• Codon: Sequence of 3 nucleotides
that code for a specific amino acid
- 61 of 64 triplets code for amino
acids
- 3 of 64 code for STOP
• Start codon – AUG
- Beginning sequence of all amino
acid chains
- Codes for amino acid methionine
• 20 total amino acids
Multiple codons can express the same
amino acid.
-Leucine  UUA, UUG, CUU,
CUC, CUA, CUG
-Codons synonymous for the
same amino acid often differ only
in the third codon position.
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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
coding strand
• RNA polymerase
5
C
DNA
G
3
A
G
T
A T C
T A
G
A G C
A
T
C G T
A
C
T
3
G C A U C G U
C
G T A G C A
T
T
A
C
A G
C T
G
A
T
A
T
3
5
unwinding
rewinding
mRNA
5
RNA polymerase
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2005 Pearson
Education, Inc. publishing as Benjamin Cummings
build
RNA
53
template strand
Bacterial chromosome
Transcription
in
Prokaryotes
Transcription
mRNA
Psssst…
no nucleus!
Cell
membrane
Cell wall
2007-2008
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Transcription in Prokaryotes
• Initiation
– RNA polymerase II binds to promoter sequence on
DNA (guided by sigma factor); strands unwind and
polymerase initiates RNA synthesis; promoter is
upstream from terminator
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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Transcription in Prokaryotes
• Elongation
– RNA polymerase
copies DNA as it
unwinds (transcription
bubble)
• ~20 base pairs at a time
300-500 bases in gene
• builds RNA 53
Simple proofreading
1 error/105 bases
 make many mRNAs
 mRNA has short life
 not worth editing!
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
reads DNA 35
Transcription in Prokaryotes
• Termination
– RNA polymerase II stops at RNA termination
sequence; RNA polymerase detaches and releases
DNA and RNA sequence is available for immediate
use
•
http://www.youtub
e.com/watch?v=1b
-bRVgqof0
2 min (no sound)
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Transcription in Eukaryotes
Transcription
Psssst…
DNA can’t
leave nucleus!
RNA Processing
Translation
Protein
2007-2008
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Transcription animation:
http://www.youtube.com/watch?hl=en&gl=US&cli
ent=mv-google&v=WsofH466lqk&nomobile=1
2.5 min overview
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Transcription: Part I - Initiation
•
Initiation complex
–
transcription factors bind to
promoter region upstream of
gene (activators/enhancers)
• 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 II to
DNA
http://www.youtube.com/watch?
v=P6Nyce-4oG42 min
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Transcription: Part II - Elongation
• RNA polymerase unzips the DNA
- Assembles RNA nucleotides - Uracil replaces Thymine
- Adds nucleotides only to the 3’ end of the growing
polymer (just like DNA polymerases)
- Creates 5’  3’ RNA molecule
• Only one DNA strand is transcribed
Transcription
rate: 40
nucleotides
per second in
eukaryotes
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Transcription: Part III - Termination
• Transcription proceeds until after the RNA polymerase
transcribes a terminator sequence in the DNA.
- In prokaryotes, RNA polymerase stops
transcription right at the end of the terminator.
• Both the RNA and DNA are then released.
- In eukaryotes, the polymerase continues for
hundreds of nucleotides past the terminator
sequence, AAUAAA (polyadenylation signal
sequence).
• At a point about 10 to 35 nucleotides past this
sequence, the mRNA is cut from the polymerase
enzyme.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
RNA Modification and Splicing
(after Transcription)
1. 5’ Cap Added
- Modified guanosine triphosphate (GTP)
- Will be attachment point for small ribosomal subunit
- Protects mRNA from degradation by hydrolytic enzymes
outside of the nucleus
2. 3’ Poly-A Tail Added
- additional 50 to 250 adenine nucleotides
- Stability. Controls movement of mRNA
- May serve to regulate gene expression
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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
3'
• add polyA tail
mRNA
A
P
5' G PP
intron = noncoding (inbetween) sequence
~10,000 bases
eukaryotic DNA
exon = coding (expressed) sequence pre-mRNA
primary mRNA
transcript
mature mRNA
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transcript
~1,000 bases
spliced mRNA
RNA 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|
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Splicing enzymes
•
•
snRNPs
–
small nuclear RNA
–
proteins
Spliceosome
–
several snRNPs
–
recognize splice site
sequence
•
cut & paste
snRNPs
snRNA
intron
exon
exon
5'
3'
spliceosome
5'
3'
lariat
5'
No,
not smurfs!
“snurps”
exon
mature mRNA
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 5'
3'
exon
3'
excised
intron
Ribozyme

RNA as ribozyme
some mRNA can even splice itself
 RNA as enzyme

Sidney Altman Thomas Cech
U ofas Benjamin
Colorado
Copyright © 2005Yale
Pearson Education, Inc. publishing
Cummings
Functional and Evolutionary Importance
of Introns
• Some genes can encode more than one kind of
polypeptide, depending on which segments are
treated as exons during RNA splicing
• Such variations are called alternative RNA splicing
• Because of alternative splicing, the number of
different proteins an organism can produce is
much greater than its number of genes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Prokaryote vs. Eukaryote genes
•
Prokaryotes
• Eukaryotes
–
DNA in cytoplasm
–
Transcription factors
–
circular chromosome
–
3 types of RNA Polymerase
–
naked DNA
–
DNA in nucleus
–
no introns
–
linear chromosomes
–
no transcription factors
–
DNA wound on histone proteins
–
introns vs. exons
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings