Lec 48: Protein Synthesis

Question 1

How is information encoded in mRNA?

Answer 1

mRNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis.

Question 2

How is the genetic code organized in relation to protein synthesis?

Answer 2

• The genetic code is degenerate (redundant). Each of the 20 common amino acids has at least one codon; many amino acids have numerous codons.
• The genetic code is non overlapping (i.e., each nucleotide is used only once), beginning with a start codon (AUG) near the 5′ end of the mRNA and ending with a termination (stop) codon (UGA, UAG, or UAA) near the 3′ end.
• The code is commaless (i.e., there are no breaks or markers to distinguish one codon rom the next).
• The start codon (AUG) determines the reading frame. Subsequent nucleotides are read in sets of three, sequentially following this codon

Question 3

What is a wobble base? How does it minimize the number of different tRNAs required to decode the mRNA?

Answer 3

A wobble base pair is a pairing between two nucleotides in RNA molecules, that does not follow Watson-Crick base pair rules. The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). Because hypoxanthine is the nucleobase of inosine, “I” is used for the former in order to maintain consistency of nucleic acid nomenclature, which otherwise follows the names of the nucleobases (e.g., “G” for both guanine and guanosine). The thermodynamic stability of a wobble base pair is comparable to that of a Watson-Crick base pair. Wobble base pairs are fundamental in RNA secondary structure and are critical for the proper translation of the genetic code.

The pairing is less specific and bases can be interchangeably recognized by the tRNA. Inosine displays the true qualities of wobble, in that if that is the first nucleotide in the anticodon than any of three bases in the original codon can be matched with the tRNA.

Question 4

What is a point mutation?

Answer 4

A single base change occurs. In proteins if this occurs in the first or third position it is often not noticed.

Question 5

What is a silent mutation?

Answer 5

A change that specifies the same amino acid

Question 6

What is a missense mutation?

Answer 6

A change that specifies a different amino acid.

Question 7

What is a nonsense mutation?

Answer 7

A change that produces a stop codon.

Question 8

What is a frameshift mutation (insertion/deletion)?

Answer 8

An addition or loss of one or more bases that affects the entire chain downstream from that point which almost always results in a nonfunctional protein. Frameshift specifically refers to an change not in a number divisible or a multiple of 3.

Question 9

Sketch the cloverleaf structure of a tRNA, and list the structural and functional features that are common to tRNAs.

Answer 9

All tRNA molecules have a similar cloverleaf structure even though their base sequences differ.

• The first loop from the 5′ end, the D loop, contains dihydrouridine.
• The middle loop contains the anticodon, which base-pairs with the codon in mRNA.
• The third loop, the TψC loop, contains both ribothymidine and pseudouridine.
• The CCA sequence at the 3′ end carries the amino acid.

Question 10

Describe the large ribosomal subunits usedfor completing the initiation complex:

Answer 10

The large ribosomal subunit binds, completing the initiation complex.

• The methionyl-tRNAiMet is bound at the P (peptidyl) site of the complex.
• The A (acceptor or aminoacyl) site of the complex is unoccupied.
• The E (ejection) site is unoccupied, and is used to remove ree tRNA from the ribosome after a peptide bond has been created between the two amino acids carried by tRNA.

Question 11

Contrast the intiation factor for translation in prokaryotes and eukaryotes:

Answer 11

Initiation factors, ATP, and GTP are required or the formation of the initiation complex.

• The initiation factors are designated as:
  
  • Prokaryotes: IF-1, IF-2, and IF-3
  • Eukaryotes: eIF-1, eIF-2, and so on. Seven or more may be present.
• The release of the initiation actors involves hydrolysis of GTP to GDP and Pi.

Question 12

Contrast the binding of mRNA in eukaryotes and prokaryotes:

Answer 12

Eukaryotes: methionyl-tRNAiMet binds to the small ribosomal subunit. The 5′ cap of the mRNA binds to the small subunit, and the first AUG codon base-pairs with the anticodon on the methionyl-tRNAiMet. The methionine that initiates protein synthesis is subsequently removed from the N-terminus of the polypeptide.

• In bacteria, the methionine that initiates protein synthesis is formylated and is carried by tRNAMet.
• Prokaryotes do not contain a 5′ cap on their mRNA. An mRNA sequence upstream from the translation start site (the Shine–Dalgarno sequence) binds to the 3′ end of 16S rRNA.

Question 13

Summarize the similarities and differences in prokaryotic and eukaryotic translation initiation:

Answer 13

see table

Question 14

Describe how amino acids are polymerized by the ribosome, including the factors and interactions that ensure the fidelity of the reaction.

Answer 14

As with any of the polymerization reactions, protein synthesis can be divided into three phases:

Initiation: where a functionally competent ribosome is assembled in the correct place on an mRNA ready to commence protein synthesis.

Elongation: whereby the correct amino acid is brought to the ribosome, is joined to the nascent polypeptide chain, and the entire assembly moves one position along the mRNA.

Termination: occurs when a stop codon is reached, there is no amino acid to be incorporated and the entire assembly dissociates to release the newly-synthesized polypeptide.

There are two rules about protein synthesis to keep in mind:

• mRNA is translated 5’ -> 3’
• Proteins are synthesized from the N-terminus to the C-terminus