Transformation-Griffith’s Expt 1928. DNA Mediates Transformation Convert IIR to IIIS By DNA?
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Transcript of Transformation-Griffith’s Expt 1928. DNA Mediates Transformation Convert IIR to IIIS By DNA?
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Transformation-Griffith’s Expt1928
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DNA Mediates Transformation
Convert IIR to IIIS By DNA?
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Avery MacLeod and McCarty ExperimentCirca 1943
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Transforming Principle
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+ means that activity is present
DNAse activity
All RNA gets degraded during enzyme preparation
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Chapter 12Translation and the Genetic Code
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Protein Structure
Proteins are complex macromolecules
composed of 20 (?)
different amino acids.
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Amino Acids Proteins are made of polypeptides. A polypeptide is a long chain of amino acids.
Amino acids have a free amino group, a free carboxyl group, and a side group (R).
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Peptide Bonds
Amino acids are joined by peptide bonds. The carboxyl group of one amino acid is
covalently attached to the amino group of the next amino acid.
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Protein Synthesis: Translation
The genetic information in mRNA molecules is translated into the amino acid sequences of
polypeptides according to the specifications of the genetic code.
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The Macromolecules of Translation
Polypeptides and rRNA molecules Euk: 28S, 18S, 5.8S, 5S
Amino-acid Activating Enzymes tRNA Molecules Soluble proteins involved in polypeptide
chain initiation, elongation, and termination
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Why does one need the ribosome to translate mRNA?
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RibosomesWhat does “S” mean? Why do sizes get bigger?
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The Nucleolus
In eukaryotes, the nucleolus is the site of rRNA synthesis and ribosome assembly
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Synthesis and Processing of the 30S rRNA Precursor in E. coli
ProKaryotenumbers
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Synthesis and Processing of the 45S rRNA Precursor in Mammals
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rRNA Genes rRNA Genes in E. coli
– Seven rRNA genes distributed among three sites on the chromosome
rRNA Genes in Eukaryotes– rRNA genes are present in hundreds to thousands
of copies– The 5.8S-18S-28S rRNA genes are present in
tandem arrays in the nucleolar organizer regions of the chromosomes.
– The 5S rRNA genes are distributed over several chromosomes.
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Transfer RNAs (tRNAs) tRNAs are adapters
between amino acids and the codons in mRNA molecules.
The anticodon of the tRNA base pairs with the codon of mRNA.
The amino acid is covalently attached to the 3’ end of the tRNA.
tRNAs often contain modified nucleosides.
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What is Inosine?
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Inosine
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tRNA Structure
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Specificity of tRNAs
tRNA molecules must have the correct anticodon sequence.
tRNA molecules must be recognized by the correct aminoacyl-tRNA synthetase.
tRNA molecules must bind to the appropriate sites on the ribosomes.
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Codon Specificity Resides in the tRNA, Not the Attached Amino Acid.
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tRNA Binding Sites on the Ribosome (Ribosme moves
like an enzyme)
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Stages of Translation
Polypeptide Chain InitiationChain ElongationChain Termination
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Translation Initiation in E. coli
30S subunit of the ribosomeInitiator tRNA (tRNAMet)mRNAInitiation Factors IF-1, IF-2, and IF-3One molecule of GTP50S subunit of the ribosome
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The Shine-Dalgarno Sequence
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Translation Initiation in Eukaryotes
The amino group of the methionine on the initiator tRNA is not formylated.
The initiation complex forms at the 5’ terminus of the mRNA, not at the Shine-Dalgarno/AUG translation start site.
The initiation complex scans the mRNA for an AUG initiation codon. Translation usually begins at the first AUG.
Kozak’s Rules describe the optimal sequence for efficient translation initiation in eukaryotes.
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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.
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Elongation of Fibroin Polypeptides (A mRNA can have multiple Ribosomes
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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.
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No tRNA exists for stop codons!
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Dissociation upon finish of protein synthesis
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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.
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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 :)
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A Triplet Code*
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A Single-Base Pair Insertion Alters the Reading Frame*
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A suppressor mutation restores the original reading frame.*
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Insertion of 3 base pairs does not change the reading
frame.*
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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.
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Deciphering the Genetic Code
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You must know single letter codes and some triplets!
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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)
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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!)
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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)
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Do all cells/animals make the sameRepertoire of tRNAs?
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The Wobble Hypothesis:Base-Pairing Involving the Third
Base of the Codon is Less Stringent.
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Base-Pairing with Inosine at the Wobble Position
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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.
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Making a (UAG) Mutation
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Translation of an amber (UAG) Mutation in the Absence of a
Suppressor tRNA
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Translation of an amber Mutation in the Presence of a Suppressor tRNA
Note it is amber su3…why?????????
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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?
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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.
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Are the proteins produced a pure reflection of the mRNA
sequence????
tRNA environment, protein modifications post-translationally
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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