Download Lecture #7 Date ______ - Phillips Scientific Methods

Chapter 17
Protein Synthesis
A Brief Review
• 1) DNA Packaging; 2) DNA Replication:
http://www.hippocampus.org/HippoCampus/
Biology;jsessionid=F30865A8DE688DE6D4
BD68F8161E8473
Overview of Transcription/Translation
(in “real time”)
• www.hippocampus.org is a great resource
for almost every subject….not just Bio!
• For this unit, specifically,Transcription and
Translation:
http://www.hippocampus.org/HippoCampu
s/Biology;jsessionid=F30865A8DE688DE6
D4BD68F8161E8473
• Other recommended sites and videos:
http://vcell.ndsu.nodak.edu/animations/
Introductory Questions #1
Name the substance that accumulates in a person’s urine causing
alkaptonuria.
2) Why did Beadle and Tatum use breadmold spores to determine
that one gene forms one polypeptide allowing for the first
metabolic pathway to be defined?
3) Transcribe & Translate the following sequence of DNA by
determining the nucleotide sequence for mRNA, the anticodon
for tRNA, and the overall amino acid sequence:
TACTCAGGACCTGCAACGATT
mRNA:
???????????????????????????????
Amino acids Sequence:
???????????????????????????????
Anticodon:
???????????????????????????????
1)
4)
5)
How does the DNA and amino acid sequences differ from a
person with sickle cell anemia and a person with normal
hemoglobin in their RBC’s? (pg. 344)
When mRNA is “processed” what is taken out (spliced)?
Protein Synthesis: Chapter 17
Bridging the gap between Genotype & Phenotypes
(proteins are thought to be that link)
Trace the Flow of Information from Gene to Protein
Key Topics:
•
•
•
•
•
•
Garrod
Beadle & Tatum
Transcription (nucleus)
Processing mRNA
Translation (cytoplasm)
Completed polypeptide (protein)
Archibald Garrod (1909)
• READ FROM TEXT: This is a synopsis
• Main Point: First to suggest that genes dictate phenotypes
through enzymes that catalyze specific chem rxns. This
led to “one gene/ one enzyme” theory
• Studied a rare genetic disorder: Alkaptonuria
• Thought to be a recessive disorder
• Tyrosine is not broken down properly into carbon dioxide
and water.
• An Intermediate substance: Alkapton (aka “Homogentisic
acid”) accumulates in the urine turning it BLACK when
exposed to air.
• An enzyme was thought to be lacking
• A genetic mutation was thought to be the cause “An
Inborn Error of Metabolism”
Garrod’s Conclusion
• A mutation in a specific gene is associated
with the absence of a specific enzyme.
• Led to the idea of:
“One Gene, One Enzyme”
• Not validated until Beadle & Tatum’s work
in the 1940’s with Neurospora (breadmold)
George Beadle & EdwardTatum
• Discovered the “One Gene, One Enzyme” Principle
• Analyzed mutations that interfered with a known
metabolic pathway
• Organism they chose to work with: Neurospora
(breadmold)
-Grows easily
-Grows as a haploid: (no homologs)
-Mutants are easily identified: Dominant allele
won’t be expressed
• Neurospora can grow easily in only: salt, sugar, &
Biotin (vitamin)
George Beadle & EdwardTatum cont’d
• Mutants-are unable to make certain organic molecules:
amino acids, lipids, etc.
• These substances are added to the media which will
allow mutants to grow successfully
• Exposed the haploid spores to x rays & UV to induce
mutations
• Haploid spores were crossed, grown in a variety of
media to determine what kind of mutation was occurring
• **They examined the effect of the mutation instead of
identifying the enzyme.
Beadle & Tatum Experiment
w/Neurospora
Beadle & Tatum’s Conclusion
“One Gene affects One Enzyme”
Later  Revised
“One Gene affects One Protein”
Later

Revised
“One Gene affects One Polypeptide Chain”
THE FLOW OF GENETIC
INFORMATION
(central dogma)
DNA → RNA → PROTEIN
• The information constituting an organism’s
genotype is carried in its sequence of bases
Protein Synthesis: overview
• Transcription:
synthesis of mRNA
under the direction of
DNA
• Translation: actual
synthesis of a
polypeptide under the
direction of mRNA
Genetic Information Written in Codons is
Translated into Amino acid Sequences
• The “words” of the DNA “language” are triplets
of bases called codons
– The codons in a gene specify the amino acid
sequence of a polypeptide
– Why triplets? *Remember there are 20 amino acids
and only 4 bases
Various Types of RNA Molecules
Gene 1
Gene 3
DNA molecule
Gene 2
DNA strand
TRANSCRIPTION
RNA
Codon
TRANSLATION
Polypeptide
Figure 10.7
Amino acid
4) How many sites are present in the ribosome? Name
the enzyme that is used to attach an amino acid to the
tRNA molecule.
• An exercise in translating the genetic code
Transcribed strand
DNA
Transcription
RNA
Start
codon
Stop
codon
Translation
Figure 10.8B
Polypeptide
The Genetic Code Dictionary
• Virtually all organisms
share the same genetic
code
• 1st codon determined
was “UUU” by
Marshal Nirenberg in
1961.
• All of the codons were
determined by the mid
1960’s
Figure 10.8A
RNA polymerase
• In transcription, the
DNA helix unzips
– RNA nucleotides line
up along one strand
of the DNA following
the base-pairing rules
– The single-stranded
messenger RNA
peels away and the
DNA strands rejoin
DNA of gene
Promoter
DNA
Initiation
Elongation
Terminator
DNA
Area shown
in Figure 10.9A
Termination
Growing
RNA
Completed RNA
Figure 10.9B
RNA
polymerase
Transcription
• Occurs in the nucleus
• RNA Polymerase is needed
-Adds nucleotides to the 3’ end only
-Eukaryotes have three types vs. Bacteria
with only one type
• Elongation occurs from 5’  3’ direction
• TATA Box : initiation site for the attachment of
RNA polymerase
• 3 Steps: Initiation  Elongation  Termination
Transcription: Initiation
• RNA Polymerase binds to the “Promoter” region
on the DNA (upstream about 25 nucleotides)
• RNA Polymerase recognizes this region because
of the “TATA” box
• Other proteins also are needed:
“Transcription factors”
Transcription produces genetic
messages in the form of RNA
RNA
polymerase
RNA nucleotide
Direction of
transcription
Template
strand of DNA
Figure 10.9A
Newly made RNA
Transcription: Elongation
• DNA is untwisted (hydrogen bonds are broken)
• About 10 base pairs are exposed
• Nucleotides are are added to the 3’ end of
the growing mRNA molecule
• Proceeds at a rate of: ~ 60 nucleotides/sec
Transcription: Termination
• Termination site is “signaled” and “read” by
RNA Polymerase
• In Eukaryotes, a polyadenylation sequence
(“AAUAAA”) is transcribed about 10-35
nucleotides before the (pre)mRNA is released
• Pre-mRNA molecule is made consisting of
“Coded” (Exons) and “Non-coded” (Introns)
regions
• Note: In Bacteria, Translation can occur as it is
released from the first transcription event
Stages of transcription
•
Transcription- The three Phases
Eukaryotic RNA is processed before
leaving the nucleus
• Noncoding
segments called
introns are spliced
out (they stay in
the nucleus)
• A cap and a tail
are added to the
ends
•
Exon Intron
Exon
Intron
Exon
DNA
Cap
RNA
transcript
with cap
and tail
Transcription
Addition of cap and tail
Introns removed
Tail
Exons spliced together
mRNA
Coding sequence
http://highered.mcgrawhill.com/sites/0072437316/student_view0/c
hapter15/animations.html#
NUCLEUS
CYTOPLASM
Figure 10.10
mRNA Structure
• 1) 5’ cap: modified guanine; protection; recognition site
for ribosomes
• 2) 3’ tail: poly(A) tail (adenine); protection;
recognition; transport
• 3) RNA splicing: involves introns & Exons
• Exons (expressed sequences) retained (exit the nucleus
to ribosome)
• Introns (intervening sequences)
-These are spliced out / spliceosome
Key Regions on Newly Transcribed
mRNA
Splicesomes w/ SNRNP’s
(Small nuclear Ribonuclearprotein-aka snurps)
Animated View of Transcription
•
http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter15/animations.html#
Translation
• Occurs in the Cytoplasm
• Key molecules and structures include:
–
–
–
–
–
mRNA
tRNA
Ribosome (30s and 40s subunits)
Free floating amino acids
Endoplasmic reticulum
3 Stages of translation
• 1) Initiation
– Brings together mRNA, tRNA and ribosome
– Begins at the start code, AUG
2) Elongation
-codon of mRNA and anticodon of tRNA
complementary bond to one another
-peptide bond fromation
-translocation-ribosome moves to next codon
3)Termination-mRNA stop codons-UAA,UAG,UGA
Translation: Overview
Transfer RNA molecules serve as
interpreters during translation
• In the cytoplasm, a
ribosome attaches to
the mRNA and
translates its
message into a
polypeptide
• The process is aided
by transfer RNAs
Amino acid attachment site
Hydrogen bond
RNA polynucleotide chain
Anticodon
Figure 10.11A
A Typical tRNA Molecule
Translation: Transfer RNA (tRNA)
mRNA from nucleus is ‘read’ along its codons by tRNA’s
anticodons at the ribosome
tRNA – has the anticodon and amino acid attached
• Each tRNA molecule has a triplet anticodon on
one end and an amino acid attachment site on the
other
Amino acid
attachment
site
Anticodon
Figure 10.11B, C
Attachment of an Amino Acid
Ribosomes Build Polypeptides
Next amino acid
to be added to
polypeptide
Growing
polypeptide
tRNA
molecules
P site
A site
Growing
polypeptide
Large
subunit
tRNA
P
A
mRNA
mRNA
binding
site
Codons
mRNA
Small
subunit
Figure 10.12A-C
• mRNA, a specific tRNA, and the ribosome
subunits assemble during initiation
Large
ribosomal
subunit
Initiator tRNA
P site
A site
Start
codon
mRNA
1
Figure 10.13B
Small ribosomal
subunit
2
Elongation adds amino acids to the
polypeptide chain until a stop codon
terminates translation
• The mRNA moves one codon at a time relative
to the ribosome
– A tRNA pairs with each codon, adding an amino
acid to the growing polypeptide
Amino acid
Polypeptide
A
site
P site
Anticodon
mRNA
1
Codon recognition
mRNA
movement
Stop
codon
New
peptide
bond
3
Translocation
2
Peptide bond
formation
Figure 10.14
New
peptide
bond
forming
Growing
polypeptide
Codons
Stage 4 Elongation
A succession of tRNAs
add their amino acids to
the polypeptide chain as
the mRNA is moved
through the ribosome,
one codon at a time.
mRNA
Polypeptide
Stop Codon
Figure 10.15 (continued)
Stage 5 Termination
The ribosome recognizes
a stop codon. The polypeptide is terminated and
released.
Translation- the Ribosome (2 subunits- large and small)
rRNA
site of mRNA codon
& tRNA anticodon
coupling
P site
holds the tRNA
carrying the growing
polypeptide chain
A site
holds the tRNA
carrying the next
amino acid to be
added to the chain
E site
discharged tRNA’s
Animated View of Transcription
•
http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter15/animations.html#
Translation- Summary
• Initiation ~ union of
mRNA, tRNA, small
ribosomal subunit;
followed by large subunit
• Elongation ~ •codon
recognition; •peptide bond
formation; •translocation
• Termination ~ ‘stop’
codon reaches ‘A’ site
• Polyribosomes: translation
of mRNA by many
ribosomes (many copies
of a polypeptide very
quickly)
Transcription & Translation in
Prokaryotes (*no nucleus)
Introductory Questions
1)
Transcribe & Translate the following sequence of DNA:
TACTCAGGACCTGCAACGATT
mRNA:
???????????????????????????????
Amino acids Sequence:
Anticodon:
2) How does the DNA and amino acid sequences differ from a
person with sickle cell anemia and a person with normal
hemoglobin in their RBC’s?
3) When mRNA is “processed” what is taken out (spliced)?
4) How many sites are present in the ribosome? Name the enzyme
that is used to attach an amino acid to the tRNA molecule.
Mutations & Errors During
Transcription & Translation
DNA Repair
• Mismatch repair:
DNA polymerase
• Excision repair:
Nuclease
• Telomere ends:
telomerase
Mutations can change the meaning of genes
• Mutations are changes in the DNA base
sequence
– These are caused by errors in DNA replication or by
mutagens
– The change of a single DNA nucleotide causes
sickle-cell disease
Mutation: Some Definitions
•
•
•
•
A heritable change in the genetic material
Mutations may be neutral, beneficial, or harmful
Mutagen: Agent that causes mutations
Spontaneous mutations: Occur in the absence of
a mutagen
Protein Synthesis and Mutation
• Mutation(Permanent, heritable DNA changes)
– Point mutation (base substitutions)
• Missense mutation
• Nonsense mutation (premature stop)
• Silent mutation
– Insertions/deletions
• Frameshift mutation
– Dramatic change in amino acids
– Run-ons, premature stops (nonsense mut.)
• The Creation of Mutation (mutagenesis)
– Spontaneous mutation
• Occurs in DNA replication (1 in 109 bp)
– Chemical mutagens
• Increases rate to 1 in 1000-100,000)
– Radiation
• X rays, gamma rays break DNA, bases
• UV light causes knots in DNA strand
• Types of mutations
NORMAL GENE
mRNA
Protein
Met
Lys
Phe
Gly
Ala
Lys
Phe
Ser
Ala
BASE SUBSTITUTION
Met
Missing
BASE DELETION
Met
Lys
Leu
Ala
His
Figure 10.16B
Normal hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
Normal hemoglobin
Sickle-cell hemoglobin
Glu
Val
Figure 10.16A
Mutation: Base Substitution (Point Mutations)
G
G
C
C
Glu
(a) Silent mutation
(d) Run-on mutation
Frameshift Mutations: Insertions/Deletions
THEBIGCATATETHERAT
THEBIGCBATATETHERAT
Figure 8.17a, d
Summary: Mutations
POINT mutations-one base pair is changed
(*may be more than one in same gene). Most common.
– Substitutions (silent, missense, nonsense)
FRAMESHIFT: Alters reading frame
– Insertions (additions)
– Deletions
• Mutagens-physical and chemical agents that
interact DNA to cause mutations—exp.: xrays
Mutations: genetic material changes in a cell