protein synthesis2005
DESCRIPTION
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PROTEIN SYNTHESISPROTEIN SYNTHESIS
PROTEIN SYNTHESIS
DNA: NUCLEIC ACID, DOUBLE STRAND, PO4, DE- OXYRIBOSE SUGAR.
BASE PAIRS (N) T=THYMINE A=ADENINE C= CYTOSINE G=GUANINE
RNA: NUCLEIC ACID, SINGLE STRAND, PO4, RIBOSE SUGAR.
BASE PAIRS (N) U = URACIL A=ADENINE C=CYTOSINE G=GUANINE
URACIL (U)base with a single-ring structure
phosphate group
sugar (ribose)
POINTS ABOUT TRANSCRIPTION
NEED RNA POLYMERASECODES FOR 20 AMINO ACIDSCODON:SERIES OF TRIPLET BASE
PAIRS.64 CODONS, 60 FOR AA, OTHERS
FOR STARTS/STOPS.INTRONS=NON-CODINGEXONS= CODING FOR RNA
PROTEIN TRANSCRIPTION
NUCLEUSRNA POLYMERASE CODES TO DNADNA TRANSCRIBES TO m-RNA INTRONS SNIPPED OUTEXONS KEPT IN CODE
unit of transcription in a DNA strandexon intron
mature mRNA transcript
poly-Atail
5’
5’ 3’
3’
(snipped out) (snipped out)
exon exonintron
cap
transcription into pre-mRNA
3’ 5’
3’5’
growing RNA transcript
5’
3’ 5’
3’
direction of transcription
RNA polymerase
sugar-phosphate backbone of one strand of nucleotides in a DNA double helix
sugar-phosphate backbone ofthe other strand of nucleotides
part of the sequence of base pairs in DNA
transcribed DNA winds up again
DNA to be transcribed unwinds
Newly forming RNA transcript
The DNA template at the assembly site
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
Binding site for mRNA
P (first
binding site for tRNA)
A (second binding site for tRNA)
TRANSCRIPTION Unwinding of gene regions of a DNA molecule
Pre mRNA Transcript Processing
mRNA rRNA tRNA
TRANSLATION
FINAL PROTEIN
Destined for use in cell or for transport
Convergence of RNAs
Synthesis of a polypetide chain at binding sites for mRNA and tRNA on the surface of an intact ribosome
Cytoplasmic pools of amino acids, tRNAs, and ribosomal subunits
Mature mRNA transcripts
protein subunits
ribosomal subunits
mature tRNA
VALINE
HISTIDINE
LEUCINE
PROLINE THREONINE
GLUTAMATE GLUTAMATE
VALINE
HISTIDINE
LEUCINE
PROLINE THREONINE
GLUTAMATE
VALINE
mRNA transcribed from the DNA
PART OF PARENTAL DNA TEMPLATE
resulting amino acid sequence
altered message in mRNA
A BASE INSERTION (RED) IN DNA
the altered amino acid sequence
ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE
ARGININE GLYCINE LEUCINE GLUTAMATELEUCINE
Overview: the roles of transcription and translation in the flow of genetic information
The triplet code
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
TranslationTranslation
Nuclearmembrane
TranscriptionTranscription
RNA ProcessingRNA Processing
TranslationTranslation
DNA
Pre-mRNA
mRNA
Ribosome
Protein
Eukaryotic Eukaryotic CellCell
TranslationTranslation
Synthesis of proteinsproteins in the cytoplasmcytoplasm
Involves the following:Involves the following:1. mRNA (codons)mRNA (codons)2. tRNA (anticodons)tRNA (anticodons)3. rRNArRNA4. ribosomesribosomes5. amino acidsamino acids
Types of RNATypes of RNA
Three types ofThree types of RNARNA:
A.A. messenger RNA (mRNA)messenger RNA (mRNA)
B.B. transfer RNA (tRNA)transfer RNA (tRNA)
C.C. ribosome RNA (rRNA)ribosome RNA (rRNA)
Remember: all produced in theRemember: all produced in the nucleusnucleus!!
A. Messenger RNA (mRNA)A. Messenger RNA (mRNA)
Carries the information for a specific proteinprotein.
Made up of 500 to 1000 nucleotides nucleotides long.
Made up of codons codons (sequence of three bases: AUG - methionine).
Each codoncodon, is specific for an amino acidamino acid.
A. Messenger RNA A. Messenger RNA (mRNA)(mRNA)
methionine glycine serine isoleucine glycine alanine stopcodon
proteinprotein
A U G G G C U C C A U C G G C G C A U A AmRNAmRNA
startcodon
Primary structure of a proteinPrimary structure of a protein
aa1 aa2 aa3 aa4 aa5 aa6
peptide bonds
codon 2 codon 3 codon 4 codon 5 codon 6 codon 7codon 1
B. Transfer RNA (tRNA)B. Transfer RNA (tRNA)
Made up of 75 to 80 nucleotides long.
Picks up the appropriate amino acid amino acid floating in the cytoplasm (amino acid amino acid activating enzymeactivating enzyme)
Transports amino acids amino acids to the mRNAmRNA.
Have anticodonsanticodons that are complementary to mRNAmRNA codonscodons.
Recognizes the appropriate codonscodons on the mRNAmRNA and bonds to them with H-bonds.
codon in mRNA
anticodon
amino acid OH
amino acidattachment site
anticodon
tRNA MOLECULE
amino acid attachment site
The structure of transfer RNA (tRNA)
B. Transfer RNA (tRNA)B. Transfer RNA (tRNA)
amino acidamino acidattachment siteattachment site
U A C
anticodonanticodon
methionine amino acidamino acid
C. Ribosomal RNA C. Ribosomal RNA (rRNA)(rRNA)
Made up of rRNArRNA is 100 to 3000 nucleotides long.
Important structural component of a ribosome.ribosome.
Associates with proteins proteins to form ribosomes.ribosomes.
RibosomesRibosomes
Large and small subunits.Large and small subunits.
Composed of rRNA (40%) rRNA (40%) and proteins proteins (60%).(60%).
Both units come together and help bind the mRNAmRNA and tRNA.tRNA.
Two sites forTwo sites for tRNAtRNAa. P siteP site (first and last tRNA will attachtRNA will attach)b. A siteA site
RibosomesOrigin Complet
e ribosome
Ribosomal subunit
rRNA components
Proteins
Cytosol (eukaryotic ribosome)
80 S 40 S60 S
18 S 5 S5.8 S25 S
C.30C.50
Chloroplasts (prokaryotic ribosome)
70 S 30 S50 S
16 S4.5 S 5 S23 S
C. 24C. 35
Mitochondrion (prokaryotic ribosome)
78 S 30 S 50 S
18 S 5 S26 S
C. 33C. 35
RibosomesRibosomes
PSite
ASite
Largesubunit
Small subunit
mRNAmRNA
A U G C U A C U U C G
TranslationTranslation
Three parts:1. initiationinitiation: start codon (AUG)2. elongationelongation:3. terminationtermination: stop codon (UAG)
Let’s make a PROTEIN!!!!PROTEIN!!!!.
TranslationTranslation
PSite
ASite
Largesubunit
Small subunit
mRNAmRNA
A U G C U A C U U C G
33
Translation
• InitiationThe 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.
34
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
35
TranslationDriven 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
36
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
InitiationInitiation
mRNAmRNA
A U G C U A C U U C G
2-tRNA
G
aa2
A U
A
1-tRNA
U A C
aa1
anticodon
hydrogenbonds codon
38
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
39
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
40
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
41
Translation
•ElongationAccompanied 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
mRNAmRNA
A U G C U A C U U C G
1-tRNA 2-tRNA
U A C G
aa1 aa2
A UA
anticodon
hydrogenbonds codon
peptide bond
3-tRNA
G A A
aa3
ElongationElongation
mRNAmRNA
A U G C U A C U U C G
1-tRNA
2-tRNA
U A C
G
aa1
aa2
A UA
peptide bond
3-tRNA
G A A
aa3
Ribosomes move over one codon
(leaves)
mRNAmRNA
A U G C U A C U U C G
2-tRNA
G
aa1
aa2
A UA
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
mRNAmRNA
A U G C U A C U U C G
2-tRNA
G
aa1aa2
A U
A
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
(leaves)
Ribosomes move over one codon
mRNAmRNA
G C U A C U U C G
aa1aa2
A
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
U G A
5-tRNA
aa5
mRNAmRNA
G C U A C U U C G
aa1aa2
A
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
U G A
5-tRNA
aa5
Ribosomes move over one codon
mRNAmRNA
A C A U G U
aa1
aa2
U
primaryprimarystructurestructureof a proteinof a protein
aa3
200-tRNA
aa4
U A G
aa5
C U
aa200
aa199
terminatorterminator or stopor stop codoncodon
TerminationTermination
Translation
•ReleaseWhen A site finally binds to a stop codon
(UGA, UAG, UAA)Stop codons bind eRF accompanied by
hydrolysis GTP to form GDP and PBinding of eRF to the stop codon alters the
specificity the peptidyl transferaseWater instead amino acid is now the
acceptor for the peptide chainProtein released from the tRNA
Translation
• The difference• Eukaryotic and prokaryotic translation can react
differently to certain antibioticsPuromycin
an analog tRNA and a general inhibitor of protein synthesis
Cycloheximideonly inhibits protein synthesis by eukaryotic ribosomes
Chloramphenicol, Tetracycline, Streptomycininhibit protein synthesis by prokaryotic ribosome
End ProductEnd Product
The end products of protein synthesis is a primary structure of a proteinprimary structure of a protein.
A sequence of amino acid amino acid bonded together by peptide bondspeptide bonds.
aa1
aa2 aa3 aa4aa5
aa200
aa199
PolyribosomePolyribosome
• Groups of ribosomes reading same mRNA mRNA simultaneously producing many proteins proteins (polypeptides).(polypeptides).
incominglargesubunit
incomingsmall subunit polypeptidepolypeptide
mRNAmRNA1 2 3 4 5 6 7
TYPES OF PROTEINS
ENZYMES/HELICASECARRIER/HEMOGLOBIN IMMUNOGLOBULIN/ANTIBODIESHORMONES/STEROIDSSTRUCTURAL/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 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
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
Coupled transcription and translation in bacteria
VALINE
HISTIDINE
LEUCINE
PROLINE THREONINE
GLUTAMATE
VALINE
original base triplet in a DNA strand
As DNA is replicated, proofreadingenzymes detect the mistake andmake a substitution for it:
a base substitution within the triplet (red)
One DNA molecule carries the original, unmutated sequence
The other DNAmolecule carries a gene mutation
POSSIBLE OUTCOMES:
OR
A summary of transcription and translation in a eukaryotic cell