NAME: MUHAMMAD HAFIZ FAZRISHAH BIN JEFFERY KIM

MATRICS NO: A179981

Short Notes (Topic): Protein Biosynthesis In Prokaryotes

• tRNA & aminoacylation

• Ribosomes
• Polypeptide synthesis
• Differences between Prokaryotes and Eukaryotes

Translation : Occurs in ribosomes

➢ Process whereby information in mRNA is translated to amino acids to form protein.

➢ Amino acids join to form protein through Polymerization

tRNA structure :

• Clover leaf in 2D and L shape in 3D

• 75-90 nucleotides
• 30-50 per cell :
o Indicates amino acid can be carried by more than 1 tRNA except for methionine (Met)
o <61 tRNAs needed due to wobble base-pairing
• Highly conserved
• Has 3 arms/loops
• Transcribed as larger precursor :
Nucleotides added, removed
Bases modified (Only in tRNA) : CCA at 3’ end and G at 5’ end

Function of tRNA :

• Converts triplet codon into specific amino acid through base pairing with anticodon
• Follows Adaptor theory (Francis Crick)
• Correct tRNA must connect to correct amino acid

tRNA charging

Aminoacyl-tRNA synthesase :

❖ Create aminoacyl-tRNA (Amino acid link to appropriate tRNA)

❖ At least 20 different ones
❖ Need ATP (Release AMP)

Amino acid react with ATP Aminoacyl-AMP + PPi Amino acid transfer to tRNA

Aminoacyl-tRNA AMP released

Ribosomes/Protein Factories

Made up of Ribosomal RNA (rRNA)

Ribosomal Proteins

➢ Have two subunits known as large subunit and small subunit

Function of Ribosome :

✓ Location/Site of Protein Synthesis

✓ Hold Mrna, aminoacyl-tRNA and translation factors in position
✓ Catalyze formation of peptide bond in protein synthesis through 23S ribosomal RNA
(rRNA)
✓ rRNA has enzyme activity

Difference of Ribosome in Prokaryote and Eukaryote:

Prokaryote (70S) Eukaryote (80S)

50S large subunit and 30S small subunit
Large subunit have 23S rRNA, 5S rRNA and 31
proteins
Small subunit have 16S rRNA (Bind to mRNA in
Shine-Dalgarno sequence) and 21 proteins
60S large subunit and 40S small subunit
Large subunit have 28S rRNA, 5S rRNA, 5.8S
rRNA and 49 proteins
Small subunit have 18S rRNA and 33 proteins

▪ Have three binding sites :

A-site : Aminoacylated tRNA (Covers large and small subunit)

P-site : Peptidyl-tRNA (Covers large and small subunit)
E-site : tRNA exit (Covers large subunit but not small subunit)

▪ Channels :

Small subunit : mRNA enter and exits

Large subunit : Polypeptide exits

Large subunit (50S) :

▪ Two RNA strands and have A,P,E site

Small subunit (30S) :

▪ Flexible, mRNA nucleotide triplet can shift to another

▪ Controls the flow of information during translation

Ribozymes/Catalytic RNA/RNAzymes
Ribosome is not a Ribozyme
o Found in introns of RNA transcript

Function : Catalyze specific biochemical reactions

Polypeptide Synthesis

• Occur in ribosome
• rRNA is synthesized during transcription :
1 will act as an enzyme, other 2 act as structural components
• tRNA function to bring amino acids to ribosome
• Information for translation to occur is from mRNA

3 Stages

Initiation

Involves 3 factors : IF1, IF2 and IF3

• IF1 : Binds to A-site on ribosome (Prevent other tRNA from binding to A-site and only bind
to P-site)
• IF2 : Bind to initiator tRNA (Bring to P-site) and bind to GTP
• IF3 : Promote dissociation of large and small ribosomal subunits

Elongation

Termination

INITIATION PHASE

❖ Involves 3 initiation factors Shine Dalgarno sequence only for

❖ First codon is always AUG and placed at P-site Prokaryote mRNA
❖ Shine Dalgarno box at -10 to AUG (Upstream of AUG)
To position the ribosome for initiation of protein synthesis
❖ End with addition of large subunit
❖ Met (First amino acid) modified to N-formylmethionine in prokaryote and not in eukaryote

➢ Met is added onto the initiator tRNA by aminoacyl-tRNA synthetase

➢ Formyl group is added then onto the methionine by methionyl-tRNA formyl transferase
➢ Formyl group is removed during termination by deformylase
➢ Met removed through post-translational modification (Not always removed as the
removal depends on the next amino acid/Amino acid with large side chain, Met would not
be removed)
If occur in bacteria, can express protein although Met not removed but protein produced
would not be like as the targeted protein

Elongation Phase

tRNA cannot enter E-site and can only enter A-site.

Have 3 factors :

✓ EF-Tu : Bind aminoacyl-tRNA and GTP

✓ EF-Ts : Displace GDP from EF-Tu
✓ EF-G : Promote translocation by GTP binding and hydrolysis
Three Stages:

• Decoding : Information present in mrna will be decoded into amino acids.

➢ Ribosome select and bind to aminoacyl tRNA at A-site (Depends whether
anticodon is complementary to mRNA codon)
➢ EF-Tu-GTP binds with aminoacyl-tRNA
➢ GTP hydrolysis causes aminoacyl-tRNA to bind to A site and release EF-Tu-GDP
➢ Then, aminoacyl-tRNA will bind to codon
➢ EF-Tu-GTP will be generated by EF-Ts by displacing GDP
• Transpeptidation : Forms peptide bond
➢ Peptidyl group in P-site is transferred to aminoacyl group in A-site
➢ Carried out by large ribosomal subunit (23S rRNA) as it has peptidyl transferase
activity
➢ Peptide bond will be formed
➢ Requires a lot of energy
• Translocation : A and P sites are transferred to P and E sites.
➢ Ribosome moved by 1 codon respectively to RNA
➢ Require EF-G-GTP (translocase) to bind to the same as EF-Tu
➢ Uncharged tRNA move to E-site from P-site
➢ Peptidyl-tRNA will move to P-site from A-site
➢ A-site will be vacant for next cycle of elongation

Rate of elongation : Slow (10-20 amino acid added per second)

▪ 1,000 nucleotides replicated per second in replication

▪ 50-100 nucleotides added per second during transcription

Reason/Why?

❖ Because it has a lot of mechanisms involved to have better control

❖ To avoid mistakes such as wrong addition of amino acid as there is no proofreading
mechanism

Termination

▪ Occurs when stop codon occupies A-site and does not bind to tRNA
▪ Process is longer than usual because aminoacyl-tRNA cannot recognise stop codon
▪ Release Factors will recognise stop codon at A-site :
➢ RF-1 : Recognise UAA n UAG
➢ RF-2 : Recognise UAA and UGA
➢ RF-3 : Helps RF-1 and RF-2 bind to ribosome through GTP hydrolysis
➢ RF-3 is a ribosome-dependent GTPase
➢ RRF : Induces ribosomal dissociation to large and small subunit together with EF-G
▪ Outcome of termination : Ribosome disassembles/dissociate , release of polypeptide
▪ In eukaryotes, only eRF-1 will recognise all stop codon
Polyribosomes

1) Polysomes (Found in Eukaryotes and Prokaryotes)

❖ Many ribosomes bound to a single mRNA (same coding region) to produce same
protein
❖ One mrna synthesises multiple copies of polypeptide
❖ Thus increases rate of protein synthesis in a short period
❖ 3’ end produces largest polypeptide chain

2) Polycistronic transcript
❖ Each gene is translated independently due to each gene having its own Shine-
Dalgarno sequence (different coding region) and produces different protein

Brief Description Of Differences Between Prokaryote and Eukaryote Translation

Prokaryote
Only 3 initiation factors
Does not have scanning process
No additional step
Initiator trna is tRNA.fmet (Met is formylated)
Has Shine-Dalgarno sequence
Does not undergo post-transcriptional process
Eukaryote
At least 11 initiation factors (eIF4)
Scanning process
Additional step (Form 43S preinitiation
complex without mRNA
Initiator trna is tRNA.met (Met is not
formylated)
Lacks Shine-Dalgarno sequence
Undergoes post-transcriptional process

Similar :

✓ Function of EF-Tu and EF-Ts in prokaryote is assumed as eEF1A and eEF1B in eukaryote
✓ Function of EF-G in prokaryote assumed by eEF2 in eukaryote
✓ eRF3 in eukaryote perform the role of bacteria RF3
✓ eRF1 operates all three stop codons