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PROTEIN SYNTHESIS
DNA: NUCLEIC ACID, DOUBLE STRAND, PO4, DEOXYRIBOSE SUGAR.
BASE PAIRS (N) T=THYMINE A=ADENINE C= CYTOSINE G=GUANINE
phosphate group
sugar (ribose)
PROTEIN TRANSCRIPTION
NUCLEUS RNA POLYMERASE CODES TO DNA DNA TRANSCRIBES TO m-RNA INTRONS SNIPPED OUT EXONS KEPT IN CODE
exon
exon
cap
poly-A tail 3
(snipped out) (snipped out)
direction of transcription
5 RNA polymerase
PROTEIN TRANSLATION
m-RNA GOES THRU RIBOSOME. RIBOSOME IS rRNA,CODE THREADS THRU RIBOSOME. AREA OF RIBOSOME BOUND TO tRNA 20 TYPES OF AA ANTICODON ON ONE END OF tRNA. AA ON OTHER END OF t-RNA AA ATTACH TO EACH OTHER IN PEPTIDE BOND FORM PROTEINS
TRANSCRIPTION
mRNA
rRNA
tRNA
TRANSLATION
Convergence of RNAs 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
VALINE LEUCINE
PROLINE
THREONINE
HISTIDINE
GLUTAMATE GLUTAMATE
PROLINE
THREONINE
VALINE
GLUTAMATE
ARGININE
GLYCINE
TYROSINE
TRYPTOPHAN
ASPARAGINE
ARGININE
GLYCINE
LEUCINE
LEUCINE
GLUTAMATE
altered message in mRNA A BASE INSERTION (RED) IN DNA the altered amino acid sequence
Overview: the roles of transcription and translation in the flow of genetic information
PEPTIDE BONDS/POLYPEPTIDES/PROTEINS
Translation
DNA
Transcription Nuclear membrane
Pre-mRNA
Eukaryotic Cell
RNA Processing
mRNA
Ribosome
Translation
Protein
Translation
Synthesis of proteins in the cytoplasm Involves the following: 1. mRNA (codons) 2. tRNA (anticodons) 3. rRNA 4. ribosomes 5. amino acids
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!
A U G G G C U C C A U C G G C G C A U A A
codon 1 codon 2
glycine
codon 3
serine
codon 4
isoleucine
codon 5
glycine
codon 6
alanine
codon 7
stop codon
protein methionine
Recognizes the appropriate codons on the mRNA and bonds to them with H-bonds.
anticodon
codon in mRNA
anticodon
OH
methionine
amino acid
U A
anticodon
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
Ribosomes
Origin Cytosol (eukaryotic ribosome) Chloroplasts (prokaryotic ribosome) Complete Ribosomal ribosome subunit 80 S 40 S 60 S rRNA components 18 S 5S 5.8 S 25 S 16 S 4.5 S 5 S 23 S 18 S 5S 26 S Proteins C.30 C.50
70 S
30 S 50 S
C. 24 C. 35
30 S 50 S
C. 33 C. 35
Ribosomes
Large subunit
P Site
A Site
mRNA
A U G
Small subunit
C U A C U U C G
Translation
Three parts: 1. initiation: start codon (AUG) 2. elongation: 3. termination: stop codon (UAG) Lets make a PROTEIN!!!!.
Translation
Large subunit
P Site
A Site
mRNA
A U G
Small subunit
C U A C U U C G
Translation
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.
33
Initiation
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
34
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
35
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
36
Initiation
aa1 aa2
2-tRNA 1-tRNA
anticodon
hydrogen bonds
U A C A U G codon
G A U C U A C U U C G A
mRNA
Translation
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
38
Elongation
Translation
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
39
Elongation
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
40
Translation
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
41
Elongation
Elongation
aa1
peptide bond
aa3 aa2
G A A
anticodon
hydrogen bonds
U A C A U G codon
G A U C U A C U U C G A
mRNA
aa1
peptide bond
aa3
aa2
1-tRNA
U A C
(leaves) 2-tRNA
3-tRNA
G A A
A U G
G A U C U A C U U C G A
mRNA
aa1
peptide bonds
aa4
aa2 aa3
4-tRNA
2-tRNA 3-tRNA
G C U
A U G
G A U G A A C U A C U U C G A A C U
mRNA
aa1
aa4
2-tRNA
G A U
(leaves) 3-tRNA
4-tRNA
G C U
A U G
G A A C U A C U U C G A A C U
mRNA
aa1
aa5
5-tRNA
U G A
3-tRNA 4-tRNA
G A A G C U G C U A C U U C G A A C U
mRNA
aa1
peptide bonds
aa5
aa2
aa3 aa4
5-tRNA
3-tRNA
U G A
4-tRNA
G A A
G C U G C U A C U U C G A A C U
mRNA
aa4
aa5
aa199
aa200
Termination
200-tRNA
A C U
mRNA
C A U G U U U A G
Translation
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
Release
Translation
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
The difference
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. aa3 aa4 aa5
aa199
aa200
aa2
aa1
Polyribosome
Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides).
mRNA
polypeptide
TYPES OF PROTEINS
ENZYMES/HELICASE CARRIER/HEMOGLOBIN IMMUNOGLOBULIN/ANTIBODIES HORMONES/STEROIDS STRUCTURAL/MUSCLE IONIC/K+,Na+ all regulate things put together critter
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
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
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 prosequence 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
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
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
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
original base triplet in a DNA strand 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
PROLINE
THREONINE
VALINE
GLUTAMATE