Polypeptide Chain Elongation
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Transcript of Polypeptide Chain Elongation
Polypeptide Chain Elongation 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.
http://www.molecularmovies.com/showcase/
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 doesDegree of DegeneracyReflect?
The Genetic Code
Initiation and termination Codons– Initiation codon: AUG– Termination codons: UAA, UAG, UGA
Degeneracy: partial and completeOrderedNearly 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 sameRepertoire 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 onehave 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 fluorescenceto figure out-codon optimize GFP?
CsCl centrifugation of DNA over time developedby Meselson and Stahl
Question 1: (0.5pts)Why does one add EtBr to CsCl gradients for the isolationof plasmid DNA?
In class question (extra credit) for Quiz #4
Question 2: (0.5pts-All or None credit)Is an 8kb supercoiled plasmid more dense than a3kb supercoiled plasmid. Yes/No (circle one)
Will an 8kb supercoiled plasmid have more EtBrbound 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 SucroseGradients 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 2BacteriaGrow FasterWhy?
Experiment 1
Experiment 2
N14 N15
only
Why would they do 2 different growth rates?
N14 N15
only
Fuse ResultsfromExpt 1 and 2
Cell Divisions
N14 N15
only
Experiment 1 observations
Watson-Crick Model
Does Expt 1 prove hybrid formation?
N14 N15
only
CriticalExperiment:
Hybrid StrandSeparationAnd CsCl centrifugation N15
ssDNAN14
ssDNA
N15
ssDNA
N15
dsDNA
Looks like control below
What aboutN14/N15 hybrid?
Movie time
RNApol IITATAA
CCATGG (Nco I site and Kozak Rule)ATG
AGGT….spliceGT……………A………polypyrimidine AG
PolyA recog sequenceAATAAA
The Reasons why ATG is a single codonand TGG is a single codon.
To Know for Exam
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