Transformation-Griffith

1
Transformation-Griffiths Expt
1928
2
DNA Mediates Transformation
Convert IIR to IIIS By DNA?
3
Avery MacLeod and McCarty Experiment
Circa 1943
4
Transforming Principle
5
(No Transcript)
6
DNAse activity
means that activity is present
All RNA gets degraded during enzyme preparation
7
Chapter 12Translation and the Genetic Code
8
Protein Structure

• Proteins are complex macromolecules
• composed of 20 (?)
• different amino acids.

9
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).

10
(No Transcript)
11
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.

12
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.

13
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

14
Why does one need the ribosome to translate mRNA?
15
Ribosomes
What does S mean?
Why do sizes get bigger?
16
The Nucleolus

• In eukaryotes, the nucleolus is the site of rRNA
  synthesis and ribosome assembly

17
Synthesis and Processing of the 30S rRNA
Precursor in E. coli
ProKaryote numbers
18
Synthesis and Processing of the 45S rRNA
Precursor in Mammals
19
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.

20
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.

21
What is Inosine?
22
(No Transcript)
23
Inosine
24
tRNA Structure
25
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.

26
Codon Specificity Resides in the tRNA, Not the
Attached Amino Acid.
27
tRNA Binding Sites on the Ribosome (Ribosme moves
like an enzyme)
28
Stages of Translation

• Polypeptide Chain Initiation
• Chain Elongation
• Chain Termination

29
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

30
(No Transcript)
31
(No Transcript)
32
The Shine-Dalgarno Sequence
33
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.
• Kozaks Rules describe the optimal sequence for
  efficient translation initiation in eukaryotes.

34
(No Transcript)
35
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.

36
(No Transcript)
37
(No Transcript)
38
(No Transcript)
39
(No Transcript)
40
(No Transcript)
41
(No Transcript)
42
Elongation of Fibroin Polypeptides (A mRNA can
have multiple Ribosomes
43
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.

44
No tRNA exists for stop codons!
45
(No Transcript)
46
Dissociation upon finish of protein synthesis
47
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.

48
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 )

49
A Triplet Code
50
A Single-Base Pair Insertion Alters the Reading
Frame
51
A suppressor mutation restores the original
reading frame.
52
Insertion of 3 base pairs does not change the
reading frame.
53
Evidence of a Triplet CodeIn 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.

54
Deciphering the Genetic Code
55
You must know single letter codes and some
triplets!
56
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)

57
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!)

58
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)

59
Do all cells/animals make the same Repertoire of
tRNAs?
60
The Wobble HypothesisBase-Pairing Involving the
Third Base of the Codon is Less Stringent.
61
Base-Pairing with Inosine at the Wobble Position
62
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.

63
Making a (UAG) Mutation
64
Translation of an amber (UAG) Mutation in the
Absence of a Suppressor tRNA
65
Translation of an amber Mutation in the Presence
of a Suppressor tRNA
Note it is amber su3why?????????
66
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?
67
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.

68
Are the proteins produced a pure reflection of
the mRNA sequence????
tRNA environment, protein modifications
post-translationally
69
To Know for Exam
RNApol II TATAA CCATGG (Nco I site and Kozak
Rule) ATG AGGT.splice GTApolypyrimidine
AG PolyA recog sequence AATAAA The Reasons why
ATG is a single codon and TGG is a single codon.