PROTEIN SYNTHESIS

PROTEIN SYNTHESIS
DNA: NUCLEIC ACID, DOUBLE STRAND, PO4, DEOXYRIBOSE 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 POLYMERASE CODES FOR 20 AMINO ACIDS CODON:SERIES OF TRIPLET BASE PAIRS. 64 CODONS, 60 FOR AA, OTHERS FOR STARTS/STOPS. INTRONS=NON-CODING EXONS= CODING FOR RNA

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

exon

unit of transcription in a DNA strand intron exon intron

exon

transcription into pre-mRNA

cap

poly-A tail 3
(snipped out) (snipped out)

5 mature mRNA transcript

sugar-phosphate backbone of one strand of nucleotides in a DNA double helix

transcribed DNA winds up again

DNA to be transcribed unwinds

Newly forming RNA transcript

The DNA template at the assembly site

sugar-phosphate backbone of the other strand of nucleotides

part of the sequence of base pairs in DNA

growing RNA transcript 5 3

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

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

protein subunits Mature mRNA transcripts ribosomal subunits mature 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

Destined for use in cell or for transport

VALINE LEUCINE

PROLINE

THREONINE

HISTIDINE

GLUTAMATE GLUTAMATE

VALINE LEUCINE HISTIDINE

PROLINE

THREONINE

VALINE

GLUTAMATE

mRNA transcribed from the DNA

PART OF PARENTAL DNA TEMPLATE

ARGININE

GLYCINE

TYROSINE

TRYPTOPHAN

ASPARAGINE

resulting amino acid sequence

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

The triplet code

TRANSCRIPTION AND TRANSLATION

C DNA. m-RNA. t-RNA. AMINO ACID ATC-GCG-TAT UAG-CGC-AUA AUC-GCG-UAU ISO-ALA-TYR

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. 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.

A. Messenger RNA (mRNA)

start codon mRNA

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

Primary structure of a protein

aa1 aa2 aa3 peptide bonds aa4 aa5 aa6

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.

anticodon

codon in mRNA

anticodon

amino acid attachment site tRNA MOLECULE

amino acid

OH

amino acid attachment site

The structure of transfer RNA (tRNA)

B. Transfer RNA (tRNA)

amino acid attachment site

methionine

amino acid

U A

anticodon

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.

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

Mitochondrion 78 S (prokaryotic ribosome)

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

3-tRNA 1-tRNA 2-tRNA

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

Ribosomes move over one codon

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

peptide bonds aa2 aa3

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

Ribosomes move over one codon

aa1

peptide bonds aa2 aa3 aa4

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

Ribosomes move over one codon

aa4

aa5
aa199
aa200

Termination

aa3 primary structure aa2 of a protein aa1

200-tRNA

terminator or stop codon

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).

incoming large subunit

mRNA

incoming small subunit

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

Coupled transcription and translation in bacteria

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

a base substitution within the triplet (red)

The other DNA molecule carries a gene mutation

VALINE LEUCINE HISTIDINE

PROLINE

THREONINE

VALINE

GLUTAMATE

A summary of transcription and translation in a eukaryotic cell