Cell-Free Protein Synthesis

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Questions and Answers

What is the purpose of wobble pairing in translation?

To increase the accuracy of amino acid sequence

To reduce the number of tRNA molecules

To allow tRNA to recognize more than one codon (correct)

To ensure Watson-Crick base pairing

Which of the following codons can pair with a tRNA with anticodon 5' GCA 3'?

5' GCC 3'

5' GCG 3'

5' GCU 3'

All of the above (correct)

What is the purpose of labelling phenylalanine with radioisotopes in a cell-free system?

To measure the concentration of amino acids

To detect the presence of ribosomes

To identify the start codon in a sequence

To trace the synthesis of proteins (correct)

How many tRNA molecules are necessary to recognize the 61 amino acid codons?

Less than 61 Signup and view all the answers

What is the significance of the third position in a codon?

It is less critical and can vary Signup and view all the answers

What is the significance of the 'start codon' AUG?

It signals the start of translation and codes for methionine Signup and view all the answers

Which of the following tRNAs could be used in the translation of the codon 5' CUG 3'?

5' GAG 3' Signup and view all the answers

What is the consequence of the genetic code being read in triplets?

There are 64 codons that code for 20 amino acids Signup and view all the answers

What is the role of Har Gobind Khorana in deciphering the genetic code?

He developed a method to synthesize RNA with a specific sequence Signup and view all the answers

What is the result of wobble pairing in translation?

Increased efficiency of translation Signup and view all the answers

What is the consequence of the genetic code being degenerate?

There are multiple codons that code for a single amino acid Signup and view all the answers

Which of the following amino acids has only one codon?

Tryptophan Signup and view all the answers

What is the significance of reading frames in the genetic code?

They determine the grouping of triplets in a sequence Signup and view all the answers

What is the significance of the first two bases in a codon?

They are critical for Watson-Crick base pairing Signup and view all the answers

What was the limitation of Nirenberg's initial approach to deciphering the genetic code?

The sequence of nucleotides in RNA could not be controlled Signup and view all the answers

What was the outcome of Har Gobind Khorana's contribution to deciphering the genetic code?

All of the above Signup and view all the answers

What is the primary function of tRNAs during translation?

To link mRNA codons with specific amino acids Signup and view all the answers

How do prokaryotic ribosomes recognize the start codon during initiation?

By recognizing a specific sequence in the 5' UTR of the mRNA Signup and view all the answers

What is the role of EF-Tu GTP during elongation?

To facilitate the binding of aminoacyl tRNA to the ribosome Signup and view all the answers

What is the outcome of the peptidyl transferase process during elongation?

The formation of a peptide bond between two amino acids Signup and view all the answers

What is the role of IF1 and IF3 during prokaryotic translation initiation?

To bind the 30S subunit and recruit mRNA Signup and view all the answers

What is the result of the binding of the large 50S subunit to the ribosome during initiation?

The formation of the complete ribosome Signup and view all the answers

What is the purpose of wobble pairing during translation?

To allow a single tRNA anticodon to recognize more than one codon Signup and view all the answers

What is the function of aminoacyl-tRNA synthetases during translation?

To charge tRNAs with the correct amino acids Signup and view all the answers

What is the function of EF-G during prokaryotic translation?

To facilitate translocation of the ribosome with GTP hydrolysis Signup and view all the answers

What occurs at the A site during prokaryotic termination?

The release factor mimics the shape of a tRNA Signup and view all the answers

How do prokaryotes and eukaryotes differ in terms of transcription?

Prokaryotes transcribe in the cytoplasm, while eukaryotes transcribe in the nucleus Signup and view all the answers

What is the function of the release factor during prokaryotic termination?

To recognize the stop codon and terminate translation Signup and view all the answers

What is the difference between prokaryotic and eukaryotic mRNA?

Prokaryotic mRNA is polycistronic, while eukaryotic mRNA is monocistronic Signup and view all the answers

What is the outcome of peptide bond formation during elongation?

A peptide bond is formed between the incoming amino acid and the growing peptide Signup and view all the answers

What occurs during translocation of the ribosome?

The ribosome moves along the mRNA with GTP hydrolysis Signup and view all the answers

What is the fate of the discharged tRNA during prokaryotic elongation?

It is released from the E site Signup and view all the answers

What is the first step in the translation process in prokaryotic cells?

Binding of the 16S rRNA with the Shine-Dalgarno sequence Signup and view all the answers

What is the function of the large 50S subunit in the initiation of translation?

It binds to the initiation factors Signup and view all the answers

What is the site of protein synthesis in eukaryotic cells?

Bound to the rough endoplasmic reticulum Signup and view all the answers

What is the role of the initiator tRNA in the initiation of translation?

It carries the first amino acid to the ribosome Signup and view all the answers

How does the RNA differ from DNA?

RNA is single-stranded, contains U instead of A and ribose instead of deoxyribose Signup and view all the answers

What is the name of the sequence that the 16S rRNA binds to in the mRNA?

Shine-Dalgarno sequence Signup and view all the answers

What is the role of the 5' cap in the translation process?

It is not involved in the translation process Signup and view all the answers

Which of the following is not a type of RNA?

Polymerase RNA Signup and view all the answers

Study Notes

Deciphering the Genetic Code

The genetic code is read in triplets, with 64 codons encoding 20 amino acids.
The code is degenerate, with multiple codons coding for the same amino acid.
One 'start codon' (AUG) signals the start of translation, and all proteins start with a methionine (Met).
Three 'stop codons' signal the termination of translation/end of protein.

Reading Frames

The genetic code can be read in different triplets, depending on the starting point.
Different reading frames produce different triplets, and each possible grouping is called a 'reading frame'.

Deciphering the Genetic Code - Historical Context

Initially, Nirenberg's lab couldn't control the order of nucleotide polymerization.
Har Gobind Khorana worked out how to synthesize RNA with a specific sequence, making it easier to assign codons to amino acids.
Khorana shared the 1968 Nobel Prize with Nirenberg and Robert W. Holley for deciphering the genetic code.

Translation: Wobble Mechanism

61 amino acid codons, but less than 61 tRNA molecules, mean some tRNAs must recognize more than one codon.
'Wobble pairing' allows a single tRNA to recognize more than one codon.
Often, the first two letters in a codon are the same, and the third base can vary.
Third position is less critical and can follow non-Watson-Crick base pairing between mRNA and tRNA.

Translation: Components

RNA and ribosomes are required for translation.
Transfer RNAs (tRNAs) function as adaptors, linking mRNA codons with specific amino acids.
Amino acids are attached to the 3' end of tRNA via an ester linkage.
The central loop of tRNA contains an anticodon, which can pair with an mRNA codon.

Prokaryotic Translation: Initiation

Initiation factors (IFs) IF1 and IF3 bind to the 30S subunit.
This complex binds mRNA, and fMet-tRNAfMet in complex with IF2-GTP enters the P site.
16S rRNA binds to the Shine-Dalgarno sequence in the mRNA to line up fMet-tRNAfMet with the AUG start codon.

Prokaryotic Translation: Elongation

Next aminoacyl tRNA binds to elongation factor EF-Tu GTP and enters the A site.
If the anticodon of the incoming tRNA is complementary to the codon, hydrolysis of GTP takes place, and EF-Tu GDP + Pi are released.
The protein is synthesized by 'lifting' the incomplete polypeptide and placing the incoming amino acid underneath.

Prokaryotic Translation: Termination

Stop codon (UAA, UAG, or UGA) is presented in the A site.
Release factor (RF1 or RF2) mimics the shape of a tRNA and enters the A site with a H2O molecule.
Peptide is hydrolyzed from the final tRNA using the H2O molecule.

Prokaryotes vs Eukaryotes

Prokaryotes: transcription occurs in the cytoplasm, polycistronic mRNA, and 30S + 50S = 70S ribosome.
Eukaryotes: transcription occurs in the nucleus, monocistronic mRNA, and 40S + 60S = 80S ribosome.
Initiation requires different mechanisms, including the interaction of 16S rRNA with the Shine-Dalgarno sequence in prokaryotes and the 5' cap interacting with the ribosome in eukaryotes.

Learning Outcomes

Outline how the genetic code was deciphered.
Explain how amino acids are encoded by groups of three nucleotide bases.
Describe the components required for translation.
Outline the process of activation of an amino acid to form an amino acyl tRNA.
Describe the 3 stages of translation in prokaryotic cells.
Identify ways in which eukaryotic translation differs from prokaryotic translation.

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Description

This quiz covers the process of cell-free protein synthesis, including the use of ribosomes, amino acids, and radiolabeling. It tests your understanding of how protein synthesis occurs in a cell-free system.