THE SYNTHESIS OF PROTEIN - TRANSLATION

We will now examine more closely how genetic information flows from mRNA to protein [Translation] in prokaryotic organisms. The sequence of codons that are present in the mRNA are translated into an amino acid sequence by another class of RNA molecules called transfer RNA (tRNA )A multi-subunit protein-RNA complex called the ribosome accomplishes this amazing feat.

First, let us examine each of the components necessary to carry out the translation process

mRNA structure:

Messenger RNA (mRNA) is transcribed from DNA by the enzyme called RNA polymerase as a single stranded, linear sequence of RNA that is complementary to one strand of the DNA. There are four types of information present in prokaryotic mRNAs, listed in order from the 5' to the 3' end of the molecule:

  1. Shine-Dalgarno sequence: This sequence correctly positions the mRNA on the ribosome.
  2. Initiation codon: this sequence of three nucleotides (AUG) indicates the start of the protein, encoding the modified amino acid N-formyl-methionine
  3. Codons: each triplet of nucleotides encodes a single amino acid.
  4. Stop codon: The triplet of UAA, UAG, or UGA does not encode an amino acid, terminating synthesis of the protein.

tRNA Structure:

A transfer RNA (tRNA) molecule consists of a single RNA strand that is only about 80 nucleotides long. There are at least 20 different tRNA molecules in the cell, one for each amino acid. The primary structure of tRNAs is simply the order of the nucleotide bases. RNA forms secondary structures, the most prevalent secondary structure is a anti-parallel helix that is similar in structure to double stranded DNA. The helical segments of the tRNA can be most easily seen by flattening the structure into one plane. This view reveals the hydrogen bonding between complementary bases. As with proteins, tRNAs can fold to form a tertiary structure that is stabilized by favorable interactions between different parts of the nucleotide chain. These interactions include additional hydrogen bonding as well as electrostatic (ionic) interactions with metal ions (K+, Mg2+).The tertiary structure is roughly L-shaped. The loop protruding from one end of the L includes the anticodon, the specialized base triplet that forms hydrogen bonds to a specific codon within the mRNA. From the other end of the L-shaped tRNA molecule protrudes its 3' end, which is the attachment site for an amino acid.

tRNA  
secondary structure
  
tertiary structure

 

Charging tRNAs:

Codon-anticodon bonding is actually the second of two recognition steps required for the accurate translation of a genetic message. It must be preceded by the creation of a correct match between tRNA and an amino acid. A tRNA that binds to a mRNA codon specifying a particular amino acid must carry only that amino acid to the ribosome. Each amino acid is joined to the correct tRNA by a specific enzyme called an aminoacyl-tRNA synthetase. There are at least 20 of these enzymes in the cell, one enzyme for each amino acid.

The active site of each type of aminoacyl-tRNA synthetase fits only a specific combination of amino acid and tRNA. The synthetase catalyzes the covalent attachment of the amino acid to its tRNA in a two-step process driven by the hydrolysis of ATP. In the first step, the enzyme activates the amino acid by attaching AMP to the carboxyl group on the amino acid. This high-energy form of the amino acid is then added to the 3' end of its tRNA, forming the aminoacyl-tRNA. This charged tRNA is released from the enzyme and is ready to deliver its amino acid to a growing polypeptide chain on a ribosome.

 

Ribosome Structure:

Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis. A ribosome is made up of two subunits, termed the large subunit and the small subunit. The ribosomal subunits are constructed of many different protein subunits and RNA molecules (ribosomal RNA (rRNA)). About 60% of the weight of each ribosome is rRNA. In prokaryotic organisms the rRNA in the small subunit is complementary to the leader sequence on the mRNA. The leader sequence is found a few nucleotides away from the initiation codon, AUG. The formation of complementary hydrogen bonds between the rRNA and the Shine-Dalgarno sequence serves to correctly position the mRNA on the ribosome. The large and small subunits join to form a functional ribosome only when they attach to a mRNA molecule.

In addition to a binding site for mRNA on the small subunit, each ribosome has three binding sites for tRNA. The peptidyl-tRNA site (P-site) that holds the tRNA carrying the growing polypeptide chain, the aminoacyl-tRNA site(A-site) holds the tRNA carrying the next amino acid to be added to the chain, and the exit site (E-site).

Chime Models:

 

30S small subunit

50S large subunit

 

 

Building a Polypeptide:

The key to translating a genetic message into a specific amino acid sequence is that each type of tRNA molecule links a particular mRNA codon with a particular amino acid. As a tRNA molecule arrives at a ribosome, it bears a specific amino acid at one end. At the other end is a nucleotide triplet called an anticodon, which binds, according to the base-pairing rules, to a complementary codon on mRNA. The ribosome then joins the amino acid to the growing poly-peptide chain.

We can divide the synthesis of a polypeptide chain, into three stages:

  1. initiation
  2. elongation
  3. termination.

All three stages require protein "factors" that aid mRNA, tRNA, and ribosomes in the translation process. For chain initiation and elongation, energy is also required. It is provided by GTP (guanosine triphosphate), a molecule closely related to ATP. The following flash animation demonstrated these three stages: