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Transformation-Griffith’s Expt. 1928. DNA Mediates Transformation. Convert IIR to IIIS By DNA?. Avery MacLeod and McCarty Experiment. Circa 1943. Transforming Principle. DNAse activity. + means that activity is present. All RNA gets degraded during enzyme preparation.

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dna mediates transformation
DNA Mediates Transformation

Convert IIR

to IIIS

By DNA?

slide6

DNAse activity

+ means that activity is present

All RNA gets degraded during enzyme preparation

protein structure

Protein Structure

Proteins are complex macromolecules

composed of 20 (?)

different amino acids.

amino acids
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).
peptide bonds
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.
protein synthesis translation

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.

the macromolecules of translation
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
ribosomes
Ribosomes

What does “S” mean?

Why do sizes get bigger?

the nucleolus
The Nucleolus
  • In eukaryotes, the nucleolus is the site of rRNA synthesis and ribosome assembly
rrna genes
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.
transfer rnas trnas
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.
specificity of trnas
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.
stages of translation
Stages of Translation
  • Polypeptide Chain Initiation
  • Chain Elongation
  • Chain Termination
translation initiation in e coli
Translation Initiation in E. coli
  • 30S subunit of the ribosome
  • Initiator tRNA (tRNAMet)
  • mRNA
  • Initiation Factors IF-1, IF-2, and IF-3
  • One molecule of GTP
  • 50S subunit of the ribosome
translation initiation in eukaryotes
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.
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.
polypeptide chain termination
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.
the genetic code

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
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 :)
evidence of a triplet code in vitro translation studies
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.
the genetic code1
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
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 points1
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)
suppressor mutations
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.
translation of an amber mutation in the presence 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 trna1
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
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
Are the proteins produced a pure reflection of the mRNA sequence????

tRNA environment, protein modifications post-translationally

slide69

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.

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