PROTEIN SYNTHESIS

PROTEIN SYNTHESISPROTEIN SYNTHESIS

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Mohammad Hanafi, MBBS., dr., MS.

Protein SynthesisProtein Synthesis

• The production (synthesis) of proteinsproteins.

• 3 phases3 phases:

1.1. TranscriptionTranscription

2.2. RNA processingRNA processing

3.3. TranslationTranslation

• Remember:Remember: DNA DNA RNA RNA ProteinProtein

DNA DNA RNA RNA ProteinProtein

Nuclearmembrane

TranscriptionTranscription

RNA ProcessingRNA Processing

TranslationTranslation

DNA

Pre-mRNA

mRNA

Ribosome

Protein

Eukaryotic Eukaryotic CellCell



DNA

mRNA

Ribosome

Protein

Prokaryotic CellProkaryotic Cell

DNA DNA RNA RNA ProteinProtein

Question:Question:

• How does RNARNA (ribonucleic acid) (ribonucleic acid) differ from DNA (deoxyribonucleic acid)DNA (deoxyribonucleic acid)?

RNARNA differs from DNADNA

1. RNARNA has a sugar ribosesugar ribose

DNADNA has a sugar deoxyribosesugar deoxyribose

2. RNARNA contains uracil (U)uracil (U)

DNADNA has thymine (T)thymine (T)

3. RNARNA molecule is single-strandedsingle-stranded

DNADNA is double-strandeddouble-stranded

1. Transcription1. Transcription

Nuclearmembrane




DNA

Pre-mRNA

mRNA

Ribosome

Protein



• The transfer of information in the nucleusnucleus from a DNADNA molecule to an RNARNA molecule.

• Only 1 1 DNADNA strand serves as the templatetemplate

• Starts at promoter DNADNA (TATA box)

• Ends at terminator DNADNA (stop)

• When complete, pre-RNApre-RNA molecule is released.

Question:Question:

• What is the What is the enzymeenzyme responsible responsible for the production of the RNA for the production of the RNA molecule?molecule?

Answer:Answer: RNA Polymerase RNA Polymerase

• Separates the DNADNA molecule by breaking the H-bonds between the bases.

• Then moves along one of the DNA strandsDNA strands and links RNARNA nucleotides together.


DNADNA

pre-mRNApre-mRNA

RNA PolymeraseRNA Polymerase

Question:Question:

• What would be the complementary RNARNA strand for the following DNADNA sequence?

• DNA 5’-GCGTATG-3’DNA 5’-GCGTATG-3’

Answer:Answer:

• DNA 5’-GCGTATG-3’DNA 5’-GCGTATG-3’

• RNA 3’-CGCAUAC-5’RNA 3’-CGCAUAC-5’

2. RNA Processing2. RNA Processing

Nuclearmembrane




DNA

Pre-mRNA

mRNA

Ribosome

Protein



• Maturation of pre-RNApre-RNA molecules.

• Also occurs in the nucleus.nucleus.

• IntronsIntrons spliced out by splicesome-enzymesplicesome-enzyme and exonsexons come together.

• End product is a mature RNA moleculemature RNA molecule that leaves the nucleusnucleus to the cytoplasm.cytoplasm.


pre-RNA molecule

intron

intronexon exon exon

exon exon exon

Mature RNA moleculeMature RNA molecule

exon exon exon

intron intron

splicesome splicesome

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.

4 nucleotides in mRNA so theoretically there are 43 (64) possible combinations of codons

Only 20 a.a.’s to encode

A. Messenger RNA (mRNA)A. Messenger RNA (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

Genetic Codes

Codons are written 5’ to 3’

Codons for the same a.a. tend to have the nucleotides in 1st and 2nd positon

AUG is unique - encodes methionine- acts as an initiation codon

Raises the possibility of 3 different reading frames depending on the start

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 enzymeamino acid activating 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.

B. Transfer RNA (tRNA)B. Transfer RNA (tRNA)

amino acidamino acidattachment siteattachment site

U A C

anticodonanticodon

methionine amino acidamino acid

tRNA CouplingAminoacyl-tRNA synthetase is required to catalyze the attachment of the specific a.a. to it’s associated tRNA

C. Ribosomal RNA (rRNA)C. Ribosomal RNA (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 (60%).proteins (60%).

• Both units come together and help bind the mRNAmRNA and tRNA.tRNA.

• Two sites forTwo sites for tRNAtRNA

a. P siteP site (first and last tRNA will attachtRNA will attach)

b. A siteA site

RibosomesRibosomes

PSite

ASite

Largesubunit

Small subunit

mRNAmRNA

A U G C U A C U U C G

RibosomesProtein formation requires orderly events to progress

Ribosomes possess binding sites to achieve this

mRNA binding site - holds mRNA strand in place

Aminoacyl-tRNA site (A-site) - holds one tRNA molecule

Peptidyl-tRNA site (P-site) - holds another tRNA molecule

Exit site (E-site) - holds a third tRNA molecule

3. Translation3. Translation

Nuclearmembrane




DNA

Pre-mRNA

mRNA

Ribosome

Protein



• Synthesis of proteinsproteins in the cytoplasmcytoplasm

• Involves the following:Involves the following:

1. mRNA (codons)mRNA (codons)

2. tRNA (anticodons)tRNA (anticodons)

3. rRNArRNA

4. ribosomesribosomes

5. amino acidsamino acids


• Three 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


InitiationInitiation

Initiation-requirements:

1. mRNA2. Ribosome3. Initiator tRNA (fMet tRNA in

prokaryotes)4. 3 Initiation factors (IF1, IF2, IF3)5. Mg2+

6. GTP (guanosine triphosphate)

Initiation-steps (e.g., prokaryotes):

1. 30S ribosome subunit + IFs/GTP bind to AUG start codon and Shine-Dalgarno sequence composed of 8-12 purine-rich nucleotides upstream (e.g., AGGAGG).

2. Shine-Dalgarno sequence is complementary to 3’ 16S rRNA.

3. Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit). All new prokaryote proteins begin with fMet (later removed).

fMet = formylmethionine (Met modified by transformylase; AUG at all other codon positions simply codes for Met)

mRNA 5’-AUG-3’ start codontRNA 3’-UAC-5’ anti-codon

4. IF3 is removed and recycled.

5. IF1 & IF2 are released and GTP is hydrolysed, catalyzing the binding of 50S rRNA subunit.

6. Results in a 70S initiation complex (mRNA, 70S, fMet-tRNA)

InitiationInitiation

mRNAmRNA


2-tRNA

G

aa2

A U

A

1-tRNA

U A C

aa1

anticodon

hydrogenbonds codon

Elongation of a polypeptide:

1. Binding of the aminoacyl tRNA (charged tRNA) to the ribosome.

2. Formation of the peptide bond.

3. Translocation of the ribosome to the next codon.

3-1. Binding of the aminoacyl tRNA to the ribosome.

• Ribosomes have two sites, P site (5’) and A site (3’) relative to the mRNA.

• Synthesis begins with fMet (prokaryotes) in the P site, and aa-tRNA hydrogen bonded to the AUG initiation codon.

• Next codon to be translated (downstream) is in the A site.

• Incoming aminoacyl-tRNA (aa-tRNA) bound to elongation factor EF-Tu + GTP binds to the A site.

• Hydrolysis of GTP releases EF-Tu, which is recycled.

• Another elongation factor, EF-Ts, removes GDP, and binds another EF-Tu + GTP to the next aa-tRNA.

• Cycle repeats after peptide bond and translocation.

mRNAmRNA


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


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


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


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)


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


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

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

Question:Question:

• The anticodon The anticodon UACUAC belongs to a belongs to a tRNAtRNA that that recognizes and binds to a particular recognizes and binds to a particular amino amino acidacid..

• What would be the What would be the DNA base code DNA base code for this for this amino acid?amino acid?

Answer:Answer:

• tRNA tRNA - UAC (anticodon)- UAC (anticodon)

• mRNAmRNA - AUG (codon)- AUG (codon)

• DNA DNA - TAC- TAC

PROTEOMEThe proteome is the final

product of genome expression and constitute all the proteins present in a cell at a particular time, It is considered as the central link between the genome and the cell.

Protein Synthesis Inhibitor:

Many of the antibiotics utilized for the treatment of bacterial infections as well as certain toxins function through the inhibition of translation. Inhibition can be affected at all stages of translation from initiation to elongation to termination.

Protein Synthesis Inhibitor:Several Antibiotic and Toxin inhibitors

of Translation:Chloramphenicol: inhibits prokaryotic

peptidyl transferaseCycloheximide: inhibits eukaryotic

peptidyl transferaseDiptheria toxin catalyzes ADP-ribosylation

of and inactivation of eEF-2Erythromycin: inhibits prokaryotic

translocation through the ribosome large subunit

Protein Synthesis Inhibitor:Fusidic acid: similar to erythromycin only by preventing

EF-G from dissociating from the large subunitNeomycin: similar in activity to streptomycinPuromycin: resembles an aminoacyl-tRNA, interferes

with peptide transfer resulting in premature termination in both prokaryotes and eukaryotes

Ricin: found in castor beans, catalyzes cleavage of the eukaryotic large subunit Rrna

Streptomycin: inhibits prokaryotic peptide chain initiation, also induces mRNA misreading

Tetracycline: inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit

THE GENETIC CODE

Genetic code is degenerate, unambiguous, none overlapping

The genetic code consists of 64 triplets of nucleotides. These triplets are called codons.

Genetic code is required to account for all 20 amino acids found in proteins. A two-letter code would have only 42 = 16 codons, which is not enough to account for all 20 amino acids, whereas a three-letter code would give 43 = 64 codons.

The 64 codons fall into groups, the members of each group coding for the same amino acid.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html

THE GENETIC CODEDegeneracy all amino acid are coded by two, three,

four or six codons except tryptophan and methionine have just a single codon each.

The code also has four punctuation codons, which indicate the points within an mRNA where translation of the nucleotide sequence should start and finish.

The initiation codon is usually 5′-AUG-3′, which also specifies methionine (so most newly synthesized polypeptides start with methionine), with a few mRNAs other codons such as 5′-GUG-3′ and 5′-UUG-3′ are used.

The three termination codons are 5′-UAG-3′, 5′-UAA-3′ and 5′-UGA-3′; these are sometimes called amber, opal and ochre, respectively.

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.glossary.9089#9301



THE GENETIC CODE

The code is not uambiguous because a given codon designates only one amino acid.

One codon, AUG serves two related functions:

It signals the start of translation.

It codes for the incorporation of the amino acid methionine (Met) into the growing polypeptide chain.

The genetic code can be expressed as either RNA codons or DNA codons:

THE GENETIC CODE

RNA codons: Occur in messenger RNA (mRNA) and

are the codons that are actually read during the synthesis of polypeptides. But each mRNA molecule acquires its sequence of nucleotides by transcription from the corresponding gene.

THE GENETIC CODE

The DNA Codons: (genes at the level of DNA):

These are the codons as they are read on the sense (5' to 3') strand of DNA. Except that the nucleotide thymidine (T) is found in place of uridine (U), they read the same as RNA codons. However, mRNA is actually synthesized using the antisense strand of DNA (3' to 5') as the template.

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PROTEIN SYNTHESIS