Transcript Lecture18 Biol302 Spring 2011

Polypeptide Chain Elongation
http://www.molecularmovies.com/showcase/
An aminoacyl-tRNA binds to the A site of the
ribosome.
The growing polypeptide chain is transferred
from the tRNA in the P site to the tRNA in the
A site by the formation of a new peptide bond.
The ribosome translocates along the mRNA
to position the next codon in the A site. At the
same time,
– The nascent polypeptide-tRNA is translocated
from the A site to the P site.
– The uncharged tRNA is translocated from the P
site to the E site.
Elongation of Fibroin
Polypeptides (A mRNA can
have multiple Ribosomes
Polypeptide Chain
Termination
Polypeptide chain termination occurs when a
chain-termination codon (stop codon) enters
the A site of the ribosome.
The stop codons are UAA, UAG, and UGA.
When a stop codon is encountered, a release
factor binds to the A site.
A water molecule is added to the carboxyl
terminus of the nascent polypeptide, causing
termination.
No tRNA exists for stop codons!
Dissociation upon finish of protein synthesis
Fig1
The Genetic Code
The genetic code is a nonoverlapping
code, with each amino acid plus
polypeptide initiation and termination
specified by RNA codons composed of
three nucleotides.
Properties of the Genetic Code
The genetic code is composed of nucleotide
triplets.
The genetic code is nonoverlapping. (?)
The genetic code is comma-free. (?)
The genetic code is degenerate. (yes)
The genetic code is ordered. (5’ to 3’)
The genetic code contains start and stop
codons. (yes)
The genetic code is nearly universal. YES :)
A Triplet Code*
A Single-Base Pair Insertion
Alters the Reading Frame*
A suppressor mutation restores
the original reading frame.*
Insertion of 3 base pairs does
not change the reading
frame.*
Evidence of a Triplet Code:
In Vitro Translation Studies
 Trinucleotides were sufficient to stimulate specific
binding of aminoacyl-tRNAs to ribosomes.
 Chemically synthesized mRNAs containing repeated
dinucleotide sequences directed the synthesis of
copolymers with alternating amino acid sequences.
 mRNAs with repeating trinucleotide sequences
directed the synthesis of a mixture of three
homopolymers.
Deciphering the Genetic Code
You must know single letter codes and some triplets!
What does
Degree of
Degeneracy
Reflect?
The Genetic Code
Initiation and termination Codons
– Initiation codon: AUG
– Termination codons: UAA, UAG, UGA
Degeneracy: partial and complete
Ordered
Nearly Universal (exceptions:
mitochondria and some protozoa)
Key Points
 Each of the 20 amino acids in proteins is specified by
one or more nucleotide triplets in mRNA. (20 amino
acids refers to what is attached to the tRNAs!)
 Of the 64 possible triplets, given the four bases in
mRNA, 61 specify amino acids and 3 signal chain
termination. (have no tRNAs!)
Key Points
 The code is nonoverlapping, with each nucleotide
part of a single codon, degenerate, with most amino
acids specified by two to four codons, and ordered,
with similar amino acids specified by related codons.
 The genetic code is nearly universal; with minor
exceptions, the 64 triplets have the same meaning in
all organisms. (this is funny)
Do all cells/animals make the same
Repertoire of tRNAs?
The Wobble Hypothesis:
Base-Pairing Involving the Third
Base of the Codon is Less Stringent.
Base-Pairing with Inosine at
the Wobble Position
Suppressor Mutations
Some mutations in tRNA genes alter the
anticodons and therefore the codons
recognized by the mutant tRNAs.
These mutations were initially detected as
suppressor mutations that suppressed the
effects of other mutations.
Example: tRNA mutations that suppress
amber mutations (UAG chain-termination
mutations) in the coding sequence of genes.
Making a (UAG) Mutation
Translation of an amber (UAG)
Mutation in the Absence of a
Suppressor tRNA
Translation of an amber Mutation in
the Presence of a Suppressor tRNA
Note it is amber su3…why?????????
Translation of an amber Mutation in
the Presence of a Suppressor tRNA
If there was a single tRNATyr gene, then could one
have a amber supressor of it?
Historical Comparisons
 Comparison of the amino acid sequence of
bacteriophage MS2 coat protein and the nucleotide
sequence of the gene encoding the protein (Walter
Fiers, 1972).
Was this first????
 Sickle-cell anemia: comparison of the sequence of
the normal and sickle-cell alleles at the amino acid
level and at the nucleotide level.
Are the proteins produced a
pure reflection of the mRNA
sequence????
tRNA environment, protein modifications post-translationally
Evolution?
Alpha and Beta chain mutants…some of them
Phylogenetic relationships
How could we use GFP fluorescence
to figure out-codon optimize GFP?
CsCl centrifugation of DNA over time developed
by Meselson and Stahl
In class question (extra credit) for Quiz #4
Question 1: (0.5pts)
Why does one add EtBr to CsCl gradients for the isolation
of plasmid DNA?
Question 2: (0.5pts-All or None credit)
Is an 8kb supercoiled plasmid more dense than a
3kb supercoiled plasmid. Yes/No (circle one)
Will an 8kb supercoiled plasmid have more EtBr
bound to it? Yes/No (circle one)
We will talk about this again in a later lecture:
But CsCl gradients are not the same thing as Sucrose
Gradients or Agarose Gel Electrophoresis.
CsCl centrifugation of DNA over time
N15 is heavier than N14-Can be resolved in CsCl
pulse-chase Experiment: Incubator with N15 containing
medium for time, then chase with N14 medium
Expt 1 grows
Slowly
Expt 2
Bacteria
Grow Faster
Why?
Why would they do 2 different growth rates?
Experiment 1
Experiment 2
N14 N15
only
N14 N15
only
Fuse Results
from
Expt 1 and 2
Cell
Divisions
N14 N15
only
Experiment 1 observations
Watson-Crick Model
N14 N15
only
Does Expt 1
prove hybrid
formation?
N15
dsDNA
N15
ssDNA
Critical
Experiment:
Hybrid Strand
Separation
And
CsCl centrifugation
Looks like
control below
What about
N14/N15 hybrid?
N14
ssDNA
N15
ssDNA
Movie time
To Know for Exam
RNApol II
TATAA
CCATGG (Nco I site and Kozak Rule)
ATG
AGGT….splice
GT……………A………polypyrimidine AG
PolyA recog sequence
AATAAA
The Reasons why ATG is a single codon
and TGG is a single codon.
STRUCTURE AND FUNCTION
OF ERYTHROPOIETIC TISSUE
The RBCs
ERYTHROPOIESIS (RBC
PRODUCTION)
Mature erythrocytes are derived
from committed erythroid proginator
cells through a series of mitotic divisions and
maturation phases.
Erythropoietin, a humoral agent produced
mainly by the kidneys stimulates
erythropoiesis by acting on committed stem
cells to induce proliferation and differentiation
of erythrocytes in the bone marrow.
ERYTHROPOIESIS
– Nucleated red cell precursors in the bone marrow
are collectively called normoblasts or
erythroblasts.
– RBCs that have matured to the non-nucleated
stage gain entry to the peripheral blood.
– Once the cells have lost their nuclei, they are
called erythrocytes.
ERYTHROPOIESIS
– Young erythrocytes that contain
residual RNA are called reticulocytes.
– Bone marrow normoblast proliferation and
maturation occurs in an orderly and well defined
sequence.
• The process involves a gradual decrease
in cell size, condensation and eventual
expulsion of the nucleus, and an increase in
hemoglobin production.
BASIC BLOOD CELL MATURATION
 Nearly all hematopoietic cells mature in the manner
shown below. For RBCs the nucleus is eventually
extruded and the cytoplasm increase correlates
with hemoglobin increase.
ERYTHROPOIESIS
GLOBIN SYNTHESIS
ASSEMBLY OF HEMOGLOBIN
Mutation:
Changing Genetic Information
ERYTHROPOIESIS
• Reticulocytes are released from the bone
marrow into the peripheral blood where they
mature into erythrocytes , usually within 24
hours.
• It is rare to see more than 1% reticulocytes in
the peripheral smear from an adult, but
common in healthy newborns.
– They can be visualized more easily by staining with
new methylene blue which allows for visualization of
the remnants of the ribosomes on the endoplasmic
reticulum.
ERYTHROPOIESIS
– Mature RBCs have a lifespan of 100-120
days and senescent RBCs are removed by
the spleen.
– 3 areas of RBC structure/metabolism are
crucial for normal erythrocyte maturation,
survival and function:
• The RBC membrane
• Hemoglobin structure and function
• Cellular energetics