Question

(a) Describe three properties of tRNA molecule.

(b) Describe two features of transmembrane region.

(c) Why a scientist might clone a protein without its transmembrane region? (2 reason)

Answer 1

Solution

1. tRNA is a molecule that is responsible for binding and carrying a specific amino acid from the specific tRNA synthetase enzyme and interacting with the translating complex of mRNA and ribosome to integrate the amino acid to the elongating polypeptide chain. for The three properties of a tRNA (transfer RNA) molecule are as follows:

• tRNA molecules are short stretches of RNA (75-90 nucleotides) consisting of 4 universal RNA bases A, U, G, C. tRNA molecules also contain rarely occurring modified bases such as dihydrouridine (D), pseudouridine (a modified uridine, denoted as   ) etc. The primary RNA chain of tRNA folds to give rise to a characteristic secondary and further folds to form tertiary structures.
• The secondary structure of tRNA molecule forms when base pairing takes place between several bases in the tRNA chain leading to the formation of four distinct stem-loops. This looks like a "clover-leaf." Five regions usually do not participate in the base pairing , they are - the CCA acceptor stem (responsible for recognizing the amino acid), the T C loop (pesudouridine containing loop), the anti-codon loop (this region interacts with the mRNA codon), the D-loop (D denotes the presence of dihydrouridine) and a variable arm.

• The third base (at the 5' end) in the tRNA anti-codon loop does not follow Watson-Crick base pairing with the cognate mRNA base, instead it forms a wobble pairing. This allows the fact that there are only 20 amino acids in a bacteria but 61 codons coding them, each of them having more than one codon leading to redundancy. The two bases apart from the third base of the anti-codon loop form Watson-Crick pairing and are conserved, however, the third base 'wobbles' and tolerates a mismatch.

2. The two features of the transmembrane region are:

• The cell membrane is fluid in nature consisting of phospholipid bilayer. The two out-facing surfaces are hydrophilic in nature and the transmembrane region is hydrophobic in nature. Any protein traversing the lipid bilayer are called transmembrane proteins. These proteins have polar regions that remain outside the membrane facing the aqueous environment and also non-polar domains consisting of amino acid residues (18-21 residues) with non-polar side chains, arranged predominantly in a-helical configurations maximizing internal hydrogen bonding to hide the polar peptide bond.
• Transmembrane proteins can be single-pass or multi-pass (more than one a-helices of the protein traversing the membrane). Some multi-pass transmembrane proteins are -barrels and are much more rigid than a-helices. The barrels are formed by sheets arranged in parallel or anti-parallel fashion. Most porin proteins are barrels. Their transmembrane regions consist of polar amino acid residues facing inside the barrel and no-polar resides facing outward interacting with the hydrophobic lipid regions of the transmembrane.

3. A scientist would clone a protein without its transmembrane region because:

• the transmembrane regions have extremely hydrophobic properties which make these proteins, when overexpressed inside the E. coli cells, be incorrectly folded to form aggregates and exist as insoluble fractions.
• the transmembrane domain containing proteins can only be extracted from overexpressing cells by using detergents that can surround the hydrophobic surfaces and allow solubilization. These detergents later hinder biological assays. The hydrophobic transmembrane domains also impart greater flexibility to the proteins and thus higher instability when not integrated to a membrane.