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PROTEIN SYNTHESIS

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protein synthesis points about transcription need rna polymerase codes for 20 amino acids codon:series of triplet base pairs. 64 codons, 60 for aa, others for starts ... – PowerPoint PPT presentation

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Title: PROTEIN SYNTHESIS


1
PROTEIN SYNTHESIS
2
POINTS ABOUT TRANSCRIPTION
  • NEED RNA POLYMERASE
  • CODES FOR 20 AMINO ACIDS
  • CODONSERIES OF TRIPLET BASE PAIRS.
  • 64 CODONS, 60 FOR AA, OTHERS FOR STARTS/STOPS.
  • INTRONSNON-CODING
  • EXONS CODING FOR RNA

3
PROTEIN TRANSCRIPTION
  • NUCLEUS
  • RNA POLYMERASE CODES TO DNA
  • DNA TRANSCRIBES TO m-RNA
  • INTRONS SNIPPED OUT
  • EXONS KEPT IN CODE

4
unit of transcription in a DNA strand
exon
intron
exon
exon
intron
3
5
transcription into pre-mRNA
poly-A tail
cap
5
3
5
3
mature mRNA transcript
5
growing RNA transcript
3
5
3
5
direction of transcription
6
PROTEIN TRANSLATION
  • m-RNA GOES THRU RIBOSOME.
  • RIBOSOME IS r-RNA,CODE THREADS THRU RIBOSOME.
  • AREA OF RIBOSOME BOUND TO tRNA
  • 20 TYPES OF AA
  • ANTICODON ON ONE END OF t-RNA.
  • AA ON OTHER END OF t-RNA
  • AA ATTACH TO EACH OTHER IN PEPTIDE BOND
  • FORM PROTEINS

7
Binding site for mRNA
P (first binding site for tRNA)
A (second binding site for tRNA)
8
(No Transcript)
9
Unwinding of gene regions of a DNA molecule
TRANSCRIPTION
Pre mRNA Transcript Processing
mRNA
rRNA
tRNA
protein subunits
Mature mRNA transcripts
ribosomal subunits
mature tRNA
Convergence of RNAs
TRANSLATION
Cytoplasmic pools of amino acids, tRNAs, and
ribosomal subunits
Synthesis of a polypetide chain at binding sites
for mRNA and tRNA on the surface of an intact
ribosome
FINAL PROTEIN
Destined for use in cell or for transport
10
VALINE
PROLINE
THREONINE
LEUCINE
HISTIDINE
GLUTAMATE
GLUTAMATE
VALINE
PROLINE
THREONINE
LEUCINE
HISTIDINE
GLUTAMATE
11
mRNA transcribed from the DNA
PART OF PARENTAL DNA TEMPLATE
resulting amino acid sequence
ARGININE
GLYCINE
TYROSINE
TRYPTOPHAN
ASPARAGINE
ARGININE
GLYCINE
LEUCINE
GLUTAMATE
LEUCINE
altered message in mRNA
A BASE INSERTION (RED) IN DNA
the altered amino acid sequence
12
Overview the roles of transcription and
translation in the flow of genetic information
13
The triplet code
14
TRANSCRIPTION AND TRANSLATION
  • C DNA. ATC-GCG-TAT
  • m-RNA. UAG-CGC-AUA
  • t-RNA. AUC-GCG-UAU
  • AMINO ACID ISO-ALA-TYR
  • PEPTIDE BONDS/POLYPEPTIDES/PROTEINS

15
(No Transcript)
16
Translation



Eukaryotic Cell
17
Translation
  • Synthesis of proteins in the cytoplasm
  • Involves the following
  • 1. mRNA (codons)
  • 2. tRNA (anticodons)
  • 3. rRNA
  • 4. ribosomes
  • 5. amino acids

18
Types of RNA
  • Three types of RNA
  • A. messenger RNA (mRNA)
  • B. transfer RNA (tRNA)
  • C. ribosome RNA (rRNA)
  • Remember all produced in the nucleus!

19
A. Messenger RNA (mRNA)
  • Carries the information for a specific protein.
  • Made up of 500 to 1000 nucleotides long.
  • Made up of codons (sequence of three bases
    AUG - methionine).
  • Each codon, is specific for an amino acid.

20
A. Messenger RNA (mRNA)
21
B. Transfer RNA (tRNA)
  • Made up of 75 to 80 nucleotides long.
  • Picks up the appropriate amino acid floating in
    the cytoplasm (amino acid activating enzyme)
  • Transports amino acids to the mRNA.
  • Have anticodons that are complementary to mRNA
    codons.
  • Recognizes the appropriate codons on the mRNA and
    bonds to them with H-bonds.

22
anticodon
codon in mRNA
anticodon
amino acid attachment site
amino acid
OH
tRNA MOLECULE
amino acid attachment site
23
The structure of transfer RNA (tRNA)
24
B. Transfer RNA (tRNA)

25
C. Ribosomal RNA (rRNA)
  • Made up of rRNA is 100 to 3000 nucleotides long.
  • Important structural component of a ribosome.
  • Associates with proteins to form ribosomes.

26
Ribosomes
  • Large and small subunits.
  • Composed of rRNA (40) and proteins (60).
  • Both units come together and help bind the mRNA
    and tRNA.
  • Two sites for tRNA
  • a. P site (first and last tRNA will attach)
  • b. A site

27
Ribosomes
Origin Complete ribosome Ribosomal subunit rRNA components Proteins
Cytosol (eukaryotic ribosome) 80 S 40 S 60 S 18 S 5 S 5.8 S 25 S C.30 C.50
Chloroplasts (prokaryotic ribosome) 70 S 30 S 50 S 16 S 4.5 S 5 S 23 S C. 24 C. 35
Mitochondrion (prokaryotic ribosome) 78 S ? 30 S ? 50 S 18 S 5 S 26 S C. 33 C. 35
28
Ribosomes
Large subunit

P Site
A Site
Small subunit
29
Translation
  • Three parts
  • 1. initiation start codon (AUG)
  • 2. elongation
  • 3. termination stop codon (UAG)
  • Lets make a PROTEIN!!!!.

30
Translation
Large subunit

P Site
A Site
Small subunit
31
Translation
  • Initiation
  • The inactive 40S and 60S subunits will bind to
    each other with high affinity to form inactive
    complex unless kept apart
  • This is achieved by eIF3, which bind to the 40S
    subunit
  • mRNA forms an initiation complex with a ribosome
  • A number of initiation factors participate in the
    process.

32
Translation
  • Cap sequence present at the 5 end of the mRNA is
    recognized by eIF4
  • Subsequently eIF3 is bound and cause the binding
    of small 40S subunit in the complexes
  • The 18S RNA present in the 40 S subunit is
    involved in binding the cap sequence
  • eIF2 binds GTP and initiation tRNA, which
    recognize the the start codon AUG
  • This complex is also bound to 40S subunit

33
Translation
  • Driven by hydrolysis of ATP, 40S complex migrate
    down stream until it finds AUG start codon
  • The large 60S subunit is then bound to the 40S
    subunit
  • It is accompanied by the dissociation of several
    initiation factor and GDP
  • The formation of the initiation complex is now
    completed
  • Ribosome complex is able to translate

34
Translation
  • Extrachromosomal mRNAs have no cap site
  • Plastid mRNA has a special ribosome binding site
    for the initial binding to the small subunit of
    the ribosome (shine-Dalgarno sequence)
  • This sequence is also found in bacterial mRNA,
    but it is not known in the mitochondria
  • In the prokaryotic, the initiation tRNA is loaded
    with N-formylmethionine
  • After peptide formation, the formyl residue is
    cleaved from the methionine

35
Initiation


anticodon
A
U
G
C
U
A
C
U
U
C
G
A
hydrogen bonds
codon
mRNA
36
Translation
  • Elongation
  • A ribosome contains two sites where the tRNAs can
    bind to the mRNA.
  • P (peptidyl) site allows the binding of the
    initiation tRNA to the AUG start codon.
  • The A (aminoacyl) site covers the second codon of
    the gene and the first is unoccupied
  • On the other side of the P site is the exit (E)
    site where empty tRNA is released

37
Translation
  • Elongation
  • The elongation begins after the corresponding
    aminoacyl-tRNA occupies the A site by forming
    base pairs with the second codon
  • Two elongation factors (eEF) play an important
    role
  • eEF1? binds GTP and guides the corresponding
    aminoacyl-tRNA to the A site, during which GTP is
    hydrolized to GDP and P.
  • The cleavage of the energy-rich anhydride bond in
    GTP enables the aminoacyl-tRNA to bind to codon
    at the A site

38
Translation
  • Elongation
  • Afterwards the GDP still bound to eEF1?, is
    exchange for GTP as mediated by the eEF1??
  • The eEF1 ?-GTP is now ready for the next cycle
  • Subsequently a peptide linkage is form between
    the carboxyl group of methionine and the amino
    group of amino acid of the tRNA bound to A site
  • Peptidyl transferase catalyzing the reaction. It
    facilitates the N-nucleophilic attack on the
    carboxyl group, whereby the peptide bond is
    formed with the released of water

39
Translation
  • Elongation
  • Accompanied by the hydrolysis of one molecule GTP
    to form GDP and P, the eEF2 facilitates the
    translocation of the ribosome along the mRNA to
    three bases downstream
  • Free tRNA arrives at site E is released, and tRNA
    loaded with the peptide now occupies the P Site
  • The third aminoacyl-tRNA binds to the vacant A
    site and a further elongation cycle can begin

40
Elongation
peptide bond
aa1
aa2


1-tRNA
2-tRNA
anticodon
U
A
C
G
A
U
A
U
G
C
U
A
C
U
U
C
G
A
hydrogen bonds
codon
mRNA
41
aa1
peptide bond
aa2


1-tRNA
U
A
C
(leaves)
2-tRNA
G
A
U
A
U
G
C
U
A
C
U
U
C
G
A
mRNA
Ribosomes move over one codon
42
peptide bonds
aa1
aa2
aa3
2-tRNA
3-tRNA
G
A
U
G
A
A
A
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
43
peptide bonds
aa1
aa2
aa3
2-tRNA
G
A
U
(leaves)
3-tRNA
G
A
A
A
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
Ribosomes move over one codon
44
peptide bonds
aa1
aa2
aa4
aa3
3-tRNA
4-tRNA
G
A
A
G
C
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
45
peptide bonds
aa1
aa2
aa3
aa4
3-tRNA
G
A
A
4-tRNA
G
C
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
Ribosomes move over one codon
46
aa5
aa4
Termination
aa199
aa200
primary structure of a protein
aa3
aa2
aa1
terminator or stop codon
200-tRNA
A
C
A
U
G
U
U
U
A
G
C
U
mRNA
47
Translation
  • Release
  • When A site finally binds to a stop codon (UGA,
    UAG, UAA)
  • Stop codons bind eRF accompanied by hydrolysis
    GTP to form GDP and P
  • Binding of eRF to the stop codon alters the
    specificity the peptidyl transferase
  • Water instead amino acid is now the acceptor for
    the peptide chain
  • Protein released from the tRNA

48
Translation
Tyr
Val
His
Met
Pro
3
CAU
UAC
GUA
CCU
5
mRNA strand
49
Translation
  • The difference
  • Eukaryotic and prokaryotic translation can react
    differently to certain antibiotics
  • ?Puromycin
  • an analog tRNA and a general inhibitor of
    protein synthesis
  • ? Cycloheximide
  • only inhibits protein synthesis by eukaryotic
    ribosomes
  • ? Chloramphenicol, Tetracycline, Streptomycin
  • inhibit protein synthesis by prokaryotic
    ribosome

50
End Product
  • The end products of protein synthesis is a
    primary structure of a protein.
  • A sequence of amino acid bonded together by
    peptide bonds.

51
Polyribosome
  • Groups of ribosomes reading same mRNA
    simultaneously producing many proteins
    (polypeptides).

52
TYPES OF PROTEINS
  • ENZYMES/HELICASE
  • CARRIER/HEMOGLOBIN
  • IMMUNOGLOBULIN/ANTIBODIES
  • HORMONES/STEROIDS
  • STRUCTURAL/MUSCLE
  • IONIC/K,Na
  • all regulate things put together critter

53
Protein Sorting
  • Vast majority of protein within the cell are
    synthesized within the cytoplasm, but the final
    sub-cellular location can be in one of a whole
    array of membrane-bound compartment
  • Protein is subjected to be sorted for special
    targeted organelles

54
Protein Sorting
  • Vast majority of protein within the cell are
    synthesized within the cytoplasm, but the final
    sub-cellular location can be in one of a whole
    array of membrane-bound compartment
  • Protein is subjected to be sorted for special
    targeted organelles
  • Plastids
  • Mitochondria
  • Peroxisomes
  • Vacuoles

55
Mitochondria
  • More than 95 of mitochondrial proteins in plant
    are encoded in the nucleus and translated in the
    cytosol
  • Proteins are generally equipped with targeting
    signals ( a signal sequence of 12-70 amino acids
    at the amino terminal)
  • Protein import occurs at translocation site
  • In most cases, protein destined for the
    mitochondrial inner membrane after transport
    through outer membrane are guided directly to the
    location by internal targeting sequence
  • Protein destined for the inner mitochondrial
    membrane contain pro-sequence that guides first
    into the mitochondrial matrix. After removal of
    the pro-sequence by processing peptidase, the
    proteins are directed by second targeting signal
    sequence into the inner membrane

56
Plastids
  • ATP is consumed for the phosphorilation of a
    protein, probably the receptor OEP86
  • The protein transport is regulated by the binding
    of the GTP to OEP86 and OEP34
  • After the protein is delivered, the pre-sequence
    is removed by a processing peptidase
  • The protein destined to thylakoid membrane are
    first delivered into stroma and then directed by
    internal targeting signal into thylakoid membrane

57
Peroxisomes
  • Small membrane-bound cytoplasmic organelle
    containing oxidizing enzymes
  • They can be found in leaf cells where they
    contain some of the enzymes of glycolytic pathway
  • All protein have to be delivered from the cytosol
  • The transport is accompanied by ATP hydrolysis
  • Targeting sequence SKL (serine-lysine-leucine)
    has been observed in C terminus, but this
    sequence is not removed after uptake

58
Vacuole
  • Proteins are transferred during their synthesis
    to the lumen of ER
  • This is aided by a signal sequence at the
    terminus of the synthesized protein, which binds
    with a signal recognition particle to a pore
    protein present in the ER membrane and thus
    directs the protein to the ER lumen
  • In such cases, ribosome is attached to the ER
    membrane during protein synthesis and the
    synthesized protein appears immediately in the ER
    lumen. It is called co-translational protein
    transport
  • This protein is then transferred from the ER by
    vesicles transfer across the golgi apparatus to
    the vacuole or are exported by secretory vesicles
    from the cell

59
Coupled transcription and translation in bacteria
60
original base triplet in a DNA strand
a base substitution within the triplet (red)
As DNA is replicated, proofreading enzymes detect
the mistake and make a substitution for it
POSSIBLE OUTCOMES
OR
One DNA molecule carries the original,
unmutated sequence
The other DNA molecule carries a gene mutation
VALINE
PROLINE
THREONINE
LEUCINE
GLUTAMATE
HISTIDINE
61
A summary of transcription and translation in a
eukaryotic cell
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