Question

translation:

Genetic code; codon. How many codons are there? How many code for amino acids? What do the others do? What is the genetic code?

tRNA; anticodon; aminoacyl-tRNA synthetase. What key role do aminoacyl-tRNA synthetases play in translation? Why is there more than one?

Ribosome structure. What are ribosomes made of? Where are they made? Why are there 3 tRNA binding sites?

Translation initiation complex

Why wouldn't a transcribed mRNA not be translated immediately?

Elongation & translocation in translation. In translocation, which parts move relative to one another, and which don’t?

Termination; release factor

Answer 1

1) Genetic code, codon: The genetic code is the instruction code that converts the genetic information present in DNA and mRNA into the set of amino acid present in protein. The set of triplet nucleotides present in DNA are first transcribed into mRNA. The nucleotides in mRNA are used to produce amino acids that are linked in a polypeptide chain. Each amino acid is coded by three nucleotides that make up the codon. There are 64 codons generated based on the combination of nucleotides. Of these, 61 codons code of 20 amino acids while three are stop codons. Stop codons do not code for any amino acids and will terminate protein synthesis. There are three termination or stop codons: UAA, UGA and UAG that terminate the polypeptide chain, as they do not code for any amino acid. Of the 61 codons, one codon can only code for one amino acid. One amino acid can however be coded by more than one codon due to Wobble third base. The first amino acid is always mostly methionine that is coded by AUG.

2) tRNA; anticodon; aminoacyl-tRNA synthetases: The tRNA molecules are adaptor molecules that bind to the codon due to complementary base-pairing at the anticodon end. Other specific amino acids are attached to the other end of tRNA. tRNA has a specific clover leaf structure formed by secondary base pairing.

Amino acyl tRNA synthetases are an enzyme that attaches the correct amino acid corresponding to the anticodon to the tRNA. They attach amino acids to their cognate tRNA molecules. Two molecules are used in formation of one amino acyl tRNA. There are 20 different tRNA synthetases present. Each of these corresponds to one amino acid. There are 20 naturally occurring amino acids present in the body. Thus, there will be more than one synthetases, which is specific for one amino acid only.

3) Ribosome structure: Translation takes place on specialized organelles called the ribosomes. Ribosomes can be present free in cytoplasm as polyribosomes or on the rough endoplasmic reticulum. Ribosome is composed of two subunits: the large subunit and the small subunit. The two subunits join on the mRNA molecule. Ribosomal subunit has protein and ribosomal RNA. Eukaryotic ribosomes are 80S made up of a 60S larger and 40S small subunit. The rRNA present is 5S, 5.8S, 16S and 28S. The prokaryotic ribosomes are 70S with a 50S large and 30S small subunit. The rRNA associated is 16S, 23S and 5S.

Eukaryotic ribosomes are synthesized and assembled in nucleolus. Ribosomal proteins enter the nucleolus. Here, the combine with the different rRNAs produced to form the mature subunits. In prokaryotes, they are synthesized in cytoplasm.

The 5’ untranslated region of mRNA has the binding site for the ribosome. In prokaryotes, it is the shine Dalgarno’s sequence. First, there is a formation of a complex on the mRNA during translation of mRNA. First, three initiation factor proteins IF1, IF2, and IF3 bind to the small subunit of the ribosome to form the pre-initiation complex. This complex along with formyl methionine carrying tRNA (fmet-tRNA) bind to mRNA to form initiation complex. This is followed by the large subunit binding to the complex to form functional ribosome. The initiation factors are released from the complex.

The larger subunit of ribosomes has three tRNA binding sites, namely A, P and E. tRNA can bind to all these sites. The A site is the amino acid site. At the A site, the aminoacyl-tRNA anticodon base pairs up with the mRNA codon. This ensures addition of correct amino acid to the protein. At the P site or polypeptide site, the amino acid is transferred from tRNA to the polypeptide chain. The E site is where the tRNA lacking the amino acid binds. The empty tRNA is then released and can be reused. Initiator rRNA is the only aminoacyl-tRNA that can directly bind the P site. All the other amino acid tRNA, even subsequent methionine, bind to A site.

4) Translation initiation complex: mRNA transcript that is formed has to undergo post transcriptional modifications before it can be translated. There is attachment of 5’cap and 3’ poly A tail. Further, in eukaryotes, mRNA is transcribed in nucleus as hnRNA. This mRNA will contain introns, which are not translated. The introns are spliced from the mRNA before the mRNA can be translated. The pre-mRNA will move from the nucleus to the cytoplasm, where proteins synthesis occur. Hence, there is a lag period before translation can occur.

5) Elongation & translocation in translation: Direction of translation is 5’ to 3’ direction. The ribosome moves along the mRNA in the 5'-to-3' direction. Elongation requires elongation factors and GTP, when the second tRNA corresponding to the second codon binds to A site. The elongation factors in E.coli are EF-Tu and EF-Ts. When the tRNA-amino acid complex binds to the A site, GTP is hydrolyzed to GDP, EF-Tu and EF-T and GDP are released to bind the next elongation factors. A peptide bond forms between the first and second amino acid by the actions of peptidyl transferase. The ribosome translocates to the next codon and the tRNA binds to E site. The A site is empty for the next tRNA. The process is repeated and the polypeptide chain grows until the ribosome encounters a stop codon.

Thus, protein translocation occurs in two steps:

i) The deacylated tRNA and the peptidyl tRNA acceptor ends move relative to the large subunit. They move from P and A sites to the E and P sites respectively. The anticodon end remain bound to smaller subunit.

ii) In next step, anticodon arms of two tRNAs move relative to the small subunit from P and A sites to the E and P sites respectively. The deacylated tRNA at end of translocation is at E site. The peptidylt tRNA is at P site while the EF-G–GDP dissociates from the ribosome.

6) Termination; release factor: As no tRNA corresponds to the stop codons, the polypeptide chain will terminate. Release factors bind to the ribosome in place of tRNA. The c terminal amino acid is cleaved from the tRNA by release factor, releasing the polypeptide. The mRNA is released from the ribosome, which is now dissociated into its two subunits.