PROTEIN SYNTHESIS

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 TRANSCRIPTIONNUCLEUSRNA 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-TATm-RNA. UAG-CGC-AUAt-RNA. AUC-GCG-UAUAMINO ACID ISO-ALA-TYR

PEPTIDE BONDS/POLYPEPTIDES/PROTEINS

TranslationTranslation

Nuclearmembrane

TranscriptionTranscription

RNA ProcessingRNA Processing


DNA

Pre-mRNA

mRNA

Ribosome

Protein

Eukaryotic Eukaryotic CellCell

TranslationTranslationSynthesis 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 RNAThree 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 proteinaa1 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 activating amino acid activating enzymeenzyme)

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

TranslationTranslationThree parts:

1. initiationinitiation: start codon (AUG)2. elongationelongation:3. terminationtermination: stop codon (UAG)

Let’s make a PROTEIN!!!!PROTEIN!!!!.


PSite

ASite

Largesubunit

Small subunit

mRNAmRNA

A U G C U A C U U C G

33

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.

34

TranslationCap sequence present at the 5’ end of the

mRNA is recognized by eIF4Subsequently eIF3 is bound and cause the

binding of small 40S subunit in the complexesThe 18S RNA present in the 40 S subunit is

involved in binding the cap sequenceeIF2 binds GTP and initiation tRNA, which

recognize the the start codon AUGThis 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

mRNAmRNAA U G C U A C U U C G

2-tRNA

G

aa2

A U

A

1-tRNA

U A C

aa1

anticodonhydrogenbonds 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


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


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


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


1-tRNA 2-tRNA

U A C G

aa1 aa2

A UA

anticodonhydrogenbonds codon

peptide bond

3-tRNA

G A A

aa3

ElongationElongation


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)


2-tRNA

G

aa1

aa2

A UA

peptide bonds

3-tRNA

G A A

aa3

4-tRNA

G C U

aa4

A C U


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)


mRNAmRNAG 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

mRNAmRNAG 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


mRNAmRNAA 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•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

Translation• The difference

• Eukaryotic and prokaryotic translation can react differently to certain antibiotics

Puromycinan 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 ProductThe 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 PROTEINSENZYMES/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

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PROTEIN SYNTHESIS