chapter 17 from gene to protein overview: the flow of genetic information the information content of...
TRANSCRIPT
Chapter 17Chapter 17From Gene to ProteinFrom Gene to Protein
Overview: The Flow of Genetic Overview: The Flow of Genetic InformationInformation
The information content of DNA is in the form of specific sequences of nucleotides
The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins
Proteins are the links between genotype and phenotype
Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation
Fig. 17-1
How does a single faulty gene result in the dramatic appearance of an albino deer?
How was the fundamental How was the fundamental relationship between genes relationship between genes and proteins discovered?and proteins discovered?
Evidence from the Study of Metabolic Evidence from the Study of Metabolic DefectsDefects
In 1909, Archibald Garrod ◦Genes dictate phenotypes through
enzymes
Symptoms of an inherited disease reflect inability to synthesize a certain enzyme
Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway
Nutritional Mutants inNutritional Mutants in Neurospora: Neurospora: Scientific InquiryScientific Inquiry
George Beadle and Edward Tatum◦ Created mutants using bread mold that
were unable to survive on minimal medium as a result of inability to synthesize certain molecules
one gene–one enzyme hypothesis◦Each gene dictates production of a specific
enzyme
Fig. 17-2a
EXPERIMENT
Growth:Wild-typecells growingand dividing
No growth:Mutant cellscannot growand divide
Minimal medium
Fig. 17-2b
RESULTSClasses of Neurospora crassa
Wild type Class I mutantsClass II mutantsClass III mutants
Minimalmedium(MM)(control)
MM +ornithine
MM +citrulline
MM +arginine(control)
Con
dit
ion
Fig. 17-2c
CONCLUSION Class I mutants(mutation in
gene A)
Class II mutants(mutation in
gene B)
Class III mutants(mutation in
gene C)Wild type
Precursor Precursor Precursor PrecursorEnzyme AEnzyme AEnzyme AEnzyme A
Ornithine Ornithine Ornithine OrnithineEnzyme BEnzyme B Enzyme BEnzyme B
Citrulline Citrulline Citrulline CitrullineEnzyme CEnzyme CEnzyme CEnzyme C
Arginine Arginine Arginine Arginine
Gene A
Gene B
Gene C
Basic Principles of Transcription & Basic Principles of Transcription & TranslationTranslation
RNA is the intermediate between genes and the proteins for which they code
Transcription is the synthesis of RNA under the direction of DNA
Transcription produces messenger RNA (mRNA)
Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA
Ribosomes are the sites of translation
• In prokaryotes, mRNA produced by transcription is immediately translated without more processing
• In a eukaryotic cell, the nuclear envelope separates transcription from translation
• Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA
• A primary transcript is the initial RNA transcript from any gene
• The central dogma is the concept that cells are governed by a cellular chain of command: DNA RNA protein
Fig. 17-3a-1
TRANSCRIPTIONDNA
mRNA
(a) Bacterial cell
Fig. 17-3a-2
(a) Bacterial cell
TRANSCRIPTIONDNA
mRNA
TRANSLATIONRibosome
Polypeptide
Fig. 17-3b-1
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNA
Fig. 17-3b-2
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNARNA PROCESSING
mRNA
Fig. 17-3b-3
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNARNA PROCESSING
mRNA
TRANSLATION Ribosome
Polypeptide
The Genetic CodeThe Genetic CodeHow are the instructions for
assembling amino acids into proteins encoded into DNA?
There are 20 amino acids, but there are only four nucleotide bases in DNA
How many bases correspond to an amino acid?
Codons: Triplets of BasesCodons: Triplets of Bases
The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words
These triplets are the smallest units of uniform length that can code for all the amino acids
During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript
During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction
Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide
Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction
Each codon specifies the addition of one of 20 amino acids
Fig. 17-4
DNAmolecule
Gene 1
Gene 2
Gene 3
DNAtemplatestrand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
Amino acid
Cracking the CodeCracking the CodeAll 64 codons were deciphered by
the mid-1960sOf the 64 triplets, 61 code for amino
acids; 3 triplets are “stop” signals to end translation
The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid
Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-5Second mRNA base
Fir
st
mR
NA
base (
5 e
nd
of
cod
on
)
Th
ird
mR
NA
base (
3 e
nd
of
cod
on
)
Evolution of the Genetic CodeEvolution of the Genetic CodeThe genetic code is nearly universal,
shared by the simplest bacteria to the most complex animals
Genes can be transcribed and translated after being transplanted from one species to another
Fig. 17-6
(a) Tobacco plant expressing a firefly gene
(b) Pig expressing a jellyfish gene
TranscriptionTranscription
fromDNA nucleic acid languagetoRNA nucleic acid language
RNARNAribose sugar N-bases
◦uracil instead of thymine◦U : A◦C : G
single strandedlots of RNAs
◦ mRNA, tRNA, rRNA, siRNA…
RNADNAtranscription
3 KINDS OF RNA3 KINDS OF RNA
RIBOSOMAL RNA (rRNA)Made in nucleolus2 subunits (large & small)Combine with proteins to
form ribosomes
3 KINDS OF RNA3 KINDS OF RNA
TRANSFER RNA (tRNA)
ANTICODON sequence matches CODON on mRNA to add correctamino acids during protein synthesis
http://www-math.mit.edu/~lippert/18.417/lectures/01_Intro/
3 KINDS OF RNA3 KINDS OF RNA
MESSENGER RNA (mRNA)carries code from DNA to ribosomes
Synthesis of an RNA TranscriptSynthesis of an RNA Transcript
The three stages of transcription:◦Initiation◦Elongation◦Termination
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-7a-4Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Initiation
33
1
RNAtranscript
5 5
UnwoundDNA
Template strandof DNA
2 Elongation
RewoundDNA
5
5 5 3 3 3
RNAtranscript
3 Termination
5
5
5 33
3Completed RNA transcript
TranscriptionTranscriptionMaking 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- pries the DNA strands apart
and hooks together the RNA nucleotides
template strand
rewinding
mRNA RNA polymerase
unwinding
coding strand
DNAC C
C
C
C
C
C
C
C CC
G
GG
G
G G
G G
G
G
GAA
AA A
A
A
A
A
A A
A
AT
T T
T
T
T
T
T
T T
T
T
U U
5
35
3
3
5build RNA 53
RNA polymerasesRNA polymerases◦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
Which gene is read?Which gene is read?Promoter region
◦ binding site before beginning of geneTranscription factors mediate the binding of RNA
polymerase and the initiation of transcription◦ TATA box binding site◦ binding site for RNA polymerase
& transcription factors
Enhancer region◦ binding site far
upstream of gene turns transcription
on HIGH
Transcription FactorsTranscription Factors
Initiation complex◦ transcription factors bind to promoter region
suite of proteins which bind to DNA hormones? turn on or off transcription
◦ trigger the binding of RNA polymerase to DNA
•Transcription
Matching bases of DNA & Matching bases of DNA & RNARNA
Match RNA bases to DNA bases on one of the DNA strands
U
A G GGGGGT T A C A C T T T T TC C C CA A
U
UU
U
U
G
G
A
A
A C CRNA
polymerase
C
C
C
C
C
G
G
G
G
A
A
A
AA
5' 3'
Eukaryotic genes have Eukaryotic genes have junk!junk!
Eukaryotic genes are not continuous◦exons = the real gene
expressed / coding DNA
◦introns = the junk inbetween sequence
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
mRNA’s require EDITING before usemRNA’s require EDITING before useMessage in NOT CONTINUOUS INTRONS are removed
Image by Riedell
mRNA splicingmRNA splicing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNAtranscript
mature mRNAtranscript
pre-mRNA
spliced mRNA
Post-transcriptional processing ◦ eukaryotic mRNA needs work after transcription◦ primary transcript = pre-mRNA◦ mRNA splicing
edit out introns ◦ make mature mRNA transcript
~10,000 bases
~1,000 bases
Splicing must be accurateSplicing must be accurateNo room for mistakes!
◦a single base added or lost throws off the reading frame
AUG|CGG|UCC|GAU|AAG|GGC|CAU
AUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
AUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGGUCCGAUAAGGGCCAU
Met|Arg|Ser|Asp|Lys|Gly|His
Met|Arg|Val|Arg|STOP|
RNA splicing enzymesRNA splicing enzymessnRNPs
exonexon intronsnRNA
5' 3'
spliceosome
exonexcisedintron
5'
5'
3'
3'
3'
lariat
exonmature mRNA
5'
snRNPs◦ small nuclear RNA◦ proteins
Spliceosome◦ several snRNPs◦ recognize splice
site sequence cut & paste gene
mRNA EDITINGmRNA EDITING
ALL ENZYMES ARE PROTEINS?
RIBOZYMES-RNA molecules that function as enzymes (In some organisms pre-RNA can remove its own introns)
PROCESSING RNA
SPLICEOSOMES
Alternative splicingAlternative splicingAlternative mRNAs produced from same gene
◦ when is an intron not an intron…◦ different segments treated as exons
A A AA
A3' poly-A tail
mRNA
5'5' cap
3'
G PPP
50-250 A’s
More post-transcriptional More post-transcriptional processingprocessing
Need to protect mRNA on its trip from nucleus to cytoplasm◦enzymes in cytoplasm attack mRNA
protect the ends of the molecule add 5 GTP cap add poly-A tail
longer tail, mRNA lasts longer: produces more protein
TranslationTranslation
fromnucleic acid languagetoamino acid language
How does mRNA code for How does mRNA code for proteins?proteins?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?
4
4
20
ATCG
AUCG
mRNA codes for proteins in mRNA codes for proteins in tripletstriplets
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
codon
The codeThe codeCode for ALL life!
◦strongest support for a common origin for all life
Code is redundant◦several codons for each amino
acid◦3rd base “wobble”- Flexible
pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon
• Start codon– AUG– methionine
• Stop codons– UGA, UAA, UAG
Why is thewobble good?
How are the codons matched How are the codons matched to amino acids?to amino acids?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
aminoacid
tRNA anti-codon
codon
5 3
3 5
3 5
UAC
MetGCA
ArgCAU
Val
Transfer RNA structureTransfer RNA structure“Clover leaf” structure
◦anticodon on “clover leaf” end◦amino acid attached on 3 end
Loading tRNA Loading tRNA Aminoacyl tRNA synthetase
◦ enzyme which bonds amino acid to tRNA◦ bond requires energy
ATP AMP bond is unstable so it can release amino acid at ribosome easily
activatingenzyme
anticodontRNATrp binds to UGG
condon of mRNA
Trp Trp Trp
mRNAA C CU GG
C=O
OHOH
H2OO
tRNATrp
tryptophan attached to tRNATrp
C=O
O
C=O
Ribosomes Ribosomes Facilitate coupling of
tRNA anticodon to mRNA codon◦organelle or enzyme?
Structure◦ribosomal RNA (rRNA) & proteins◦2 subunits
large small E P A
Ribosomes Ribosomes
Met
5'
3'
UUA C
A G
APE
A site (aminoacyl-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
Protein synthesis 2
Building a polypeptideBuilding a polypeptideInitiation
◦ brings together mRNA, ribosome subunits, initiator tRNA
Elongation◦ adding amino acids based on
codon sequence
Termination◦ end codon 123
Leu
Leu Leu Leu
tRNA
Met MetMet Met
PE AmRNA5' 5' 5' 5'
3' 3' 3'3'
U UA AAACC
CAU UG G
GUU
A AAAC
CC
AU UG GGU
UA
AAAC
CC
AU UG GGU U
A AACCA U UG G
G AC
ValSer
AlaTrp
releasefactor
AA A
CCU UGG 3'
How translation works
Fig. 17-18-4
Amino endof polypeptide
mRNA
5
3E
Psite
Asite
GTP
GDP
E
P A
E
P A
GDPGTP
Ribosome ready fornext aminoacyl tRNA
E
P A
Protein targeting Protein targeting
Signal peptide◦address label
Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc…start of a secretory pathway
Protein Protein Synthesis in Synthesis in ProkaryotesProkaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Psssst…no nucleus!
Prokaryote vs. Eukaryote Prokaryote vs. Eukaryote genesgenes
Prokaryotes◦ DNA in cytoplasm◦ circular
chromosome◦ naked DNA◦ no introns
Eukaryotes◦ DNA in nucleus◦ linear chromosomes◦ DNA wound on
histone proteins◦ introns vs. exons
eukaryoticDNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequenceintrons
come out!
Transcription & translation are simultaneous in bacteria ◦DNA is in
cytoplasm◦no mRNA
editing ◦ribosomes
read mRNA as it is being transcribed
Translation in ProkaryotesTranslation in Prokaryotes
Translation: prokaryotes vs. Translation: prokaryotes vs. eukaryoteseukaryotes
Differences between prokaryotes & eukaryotes◦time & physical separation between
processes takes eukaryote ~1 hour
from DNA to protein◦no RNA processing
SEE PROCESSING VIDEO
COMPLETING PROTEINSCOMPLETING PROTEINSPOLYRIBOSOMES (POLYSOMES)
◦Numerous ribosomes translate same mRNA at same time
◦3-D folding (1’, 2’, 3’ structure)◦Chaparonins
POST-TRANSLATIONAL MODIFICATIONS◦ Some amino acids modified by addition of
sugars, lipids, phosphate groups, etc◦ Enzymes can modify ends, cleave into pieces
join polypeptide strands (4’ structure)
Ex: Made as proinsulin then cut
Final insulin hormonemade of two chains connected by disulfide bridges
http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin.html
Can you tell the story?
DNA
pre-mRNA
ribosome
tRNA
aminoacids
polypeptide
mature mRNA
5' GTP cap
poly-A tail
large ribosomal subunit
small ribosomal subunit
aminoacyl tRNAsynthetase
E P A
5'
3'
RNA polymerase
exon intron
tRNA
MutationsMutationsMutation- change in genetic
material of a cell◦Point Mutations- chemical changes in
just one base pair Base-pair substitutions Base-pair insertions or deletions
Base Pair SubstitutionsBase Pair SubstitutionsBase-pair substitution-
replacement of one nucleotide and its partner, with another pair of nucleotides
silent mutation no amino acid change redundancy in code
missense Codes for different amino
acid nonsense
Codes for a stop codon
Slide from Explore Biology by Kim Foglia
Point mutation leads to Sickle cell Point mutation leads to Sickle cell anemiaanemia
What kind of mutation?
Slide from Explore Biology by Kim Foglia
Base pair substitution that caused missense mutation
Sickle cell anemiaSickle cell anemia
Slide from Explore Biology by Kim Foglia
Base Pair Insertions or Base Pair Insertions or DeletionsDeletions
Frameshift - shift in the reading frame
◦ Insertions- adding base(s)◦ Deletions- losing base(s)
Frameshift mutation- occur whenever number of nucleotides inserted or deleted are not multiples of three
changes everything “downstream”
◦ More damaging at beginning of gene than at end
Slide modified from: Explore Biology by Kim Foglia
MutagensMutagensMutagens- physical or chemical
agents that interact with DNA in ways that cause mutations◦Ex. 1920’s x-rays discovered to
cause genetic changes in fruit flies◦Application today: preliminary
screening of chemicals to identify carcinogens
WHAT IS A “GENE”?WHAT IS A “GENE”?Mendel’s factors determine phenotype
T.H. Morgan- genes located on specific chromosomes
Beadle and Tatum’s “one gene-one enzyme”
Became “One gene-one polypeptide”
- Some proteins made of more than one polypeptide chainEx: hemoglobin has 4 polypeptide chains
Now: “one gene – one polypeptide or RNA” - Not all genes code for proteins
DNA TechnologyGel
electrophoresis- technique that uses gel as a molecular sieve to separate nucleic acids or proteins based on their size, electrical charge, and other physical properties
How does it work?Restriction enzymes- enzymes that cut
DNA molecules at a limited number of specific locations◦ In nature protect cell from foreign DNA
All copies of a particular DNA molecule always yield the same set of restriction fragments when exposed to the same restriction enzyme
http://learn.genetics.utah.edu/content/labs/gel/