There are many types of RNAs, but three types of it are described here: 1. Ribosomal RNA (rRNA) 2. Messenger RNA (mRNA) 3. Transfer RNA (tRNA).
Type # 1. The Ribosomal RNA (rRNA):
The non-genetic RNAs are synthesized on the DNA template and are present in the nucleolus and cytoplasm. Therefore, the base sequences of rRNA and part of DNA where they are synthesized are complementary. In prokaryotes rRNA is formed on a part of DNA called ribosomal DNA, while in eukaryotes these are formed in nucleolus containing the nuclear DNA.
The rRNAs are found in ribosomes and accounts for 40-60% of dry weight. In general, it represents about 80% of total RNA of the cell. The ribosome consists of proteins and RNA. The ribosomes are of different types such as 8OS (found in eukaryotes) and 55S (found in mitochondria of vertebrates).
The 70S ribosomes of prokaryotes are made up of two subunits, 5OS and 30S. The SOS subunit contains 23S and 5S rRNA, whereas the 30S subunit consists of 16S rRNA.
The 8OS ribosome consists of 60S and 40S subunit. The rRNA types in both the subunits of plants differ from that of animals (Table 5.6).
The rRNA is a single stranded molecule which is twisted at certain points to form helical regions. In the helical region most of the base pairs are complementary and linked by hydrogen bonds. The uncoiled single stranded regions lack the complementary bases. Therefore, in rRNA the ratio of purine: pyrimidine is not equal. The rRNA exists in a living cell for about two generations.
Type # 2. The Messenger RNA (mRNA):
The mRNA is transcribed on the DNA template and, therefore, carries the genetic information of DNA. For the first time, Francis Jacob and Jacques Monod (1961) proposed the name mRNA for bearing the transcripts of DNA for protein synthesis on ribosomes.
The total population of mRNA in a cell varies from 5 to 10% of the total cellular RNA because the species of mRNA are short lived as these are broken into ribonucleotides by the enzyme ribonuclease. In E.coli some of the mRNAs remain alive only for about two minutes. Therefore, the cell does not contain high amount of mRNA at a time. In contrast, the mRNAs of eukaryotes are metabolically stable.
The size of mRNA varies. The smallest protein contains about 50 amino acids (50 × 3=150 nucleotides needed for monocistronic mRNA molecules). Typically protein has 300-600 amino acids (900- 1,800 nucleotide long mRNA). In prokaryotes the polycistronic mRNA is more common than monocistronic mRNA and contains 3000-8000 nucleotides. Polycistronic mRNA contains usually 10 bases long intercistronic sequences called spacers.
The sedimentation coefficient of mRNA is 8S and average molecular weight ranges from 500,000 to 1,00,000. Since they represent a gene, their length and molecular weight change because a gene contains 100 to 1,500 nucleotides. The mRNAs are transcribed by genes, hence individual mRNA represents a single gene.
Therefore, in a cell there will be as much mRNAs as genes, and every mRNA differs from each other. Taylor (1979) has reviewed the isolation of eukaryotic mRNAs. Kozak (1983) has given a comparative account of initiation of protein synthesis in prokaryotes, eukaryotes and the organelles.
Initiation of synthesis of first polypeptide chain of a polycistronic mRNA may begin hundreds of nucleotides from the 5′ end. The section of non-translated RNA before coding region is called leader. Un-translated sequences are usually formed at both 5′ and 3′ ends.
As the mRNAs always remain in single stranded form, it may disrupt the biological activity after being coiled. However, the coils lack complementary bases. Kozak (1991) has discussed the structural features in eukaryotic mRNAs that modulate the initiation of translation.
The structure of prokaryotic and eukaryotic mRNA is shown in Fig 5.12 (A-C) and discussed below:
(i) The 5′ Cap:
In most of the eukaryotes and animal viruses, 5′ end of mRNA contains a cap which is formed after methylation of any of four nucleotides. For example, an mRNA contains m7G (5′) ppp (5′)N where m7G is the methylguanosine and (5′)ppp(5′) represents a 5-5′ triphosphate linked to a base (N) at 5′ end. The mRNA binds to ribosome with the help of this cap. Therefore, it governs protein synthesis.
The bacterial mRNA does not contain 5′ cap. Instead they contain a specific ribosome binding site about six nucleotide long which occurs at several places in the mRNA molecules. These are located at 4 nucleotide upstream from AUC. In bacterial mRNA there may be multiple ribosome binding sites called Shine- Dalgarno sequences in the interior of an mRNA chain, each resulting in synthesis of a different protein.
(ii) The Non-coding Regions:
There are two non-coding regions first followed by the cap and the second followed by the termination codon. The non-coding region (NCI) is about 10-100 nucleotides long and rich in A and G residues, whereas the NC2 is 50-150 nucleotides long and contains an AAUAAA residues. Both the non-coding regions do not translate protein.
(iii) The Initiation Codon:
Both in prokaryotes and eukaryotes the initiation codon (AUG) is present which starts protein synthesis. Bacterial ribosomes, unlike the eukaryotic ribosomes, directly bind to start codons in the interior of mRNA to initiate protein synthesis.
(iv) The Coding Region:
It is the most important region of mRNA which is about 1,500 nucleotides long. This region translates a long chain of protein after attaching with several ribosomes. The combination of mRNA strand with several ribosomes is called polyribosome.
Therefore, the bacterial mRNAs are commonly called polycistronic mRNA i.e. they encode multiple proteins that are separately translated from the same mRNA molecule. The eukaryotic mRNAs are typically monocistronic i.e. only one species of polypeptide chain is translated per mRNA molecule.
(v) The Termination Codon:
The termination codon is required to give the signal to stop protein synthesis. In eukaryotes the termination codons are UAA, UAG or UGA that terminates the translation process i.e. the process of protein synthesis.
(vi) The Poly (A) Sequence:
The NC2 is followed by poly (A) sequence in the eukaryotic mRNA. The prokaryotic mRNAs lack poly (A). The polyadenylate or poly (A) sequences of 200- 250 nucleotides are present at 3’OH end of mRNA. Poly (A) sequences are added when mRNA is present inside the nucleus.The function of poly (A) sequence in translation is unknown.
Type # 3. The Transfer RNA (tRNA) or Soluble RNA (sRNA):
Twenty different amino acids required for protein synthesis are present in cytoplasm. Before joining an appropriate amino acid together to form protein, they are activated by attaching to the RNA. Requirement of energy for activation is met from ATP.
The RNA which is capable to transfer an amino acid from amino acid pool, possesses capacity to combine with only one amino acid in the presence of an enzyme, aminoacyl tRNA synthetase, and recognises the codon of mRNA, is called tRNA or sRNA.
For each amino acid there is different tRNA. It is likely that 20 different tRNAs are present in cytoplasm. However in several cases more than one type of tRNA for each amino acid is present. Therefore, there are more tRNAs than the amino acids. For example, about 100 types of tRNAs are found in bacterial cell.
The mRNA, contain codes of each of three nucleotides called codon which specifies a single amino acid. The tRNA molecules read the coded message on mRNA. Therefore, the tRNA molecules act as interpreter of genetic code.
(i) Structure of tRNA:
The tRNA is dissolved in cytoplasm and is too small to be precipitated even at 1,00,000 g. Its molecular weight ranges from 25,000 to 30,000 D and sedimentation coefficient is 3.85. It accounts for 10-20% of the total cytoplasmic RNA. These are synthesized in the nucleus on a DNA template by only 0.025% of total DNA at the end of cleavage stage.
As the tRNA synthesis is over on DNA template, a part of ribonucleotide (5’CCA3′) is added to the 3′ end of each molecule regardless of amino acid affinity, by an enzyme tRNA phosphorylase. It is the special feature of tRNA as compared to mRNA and rRNA.
The tRNA molecules contain about 70-93% nucleotides arranged in a single strand at 5 → 3′ ends. This sstRNA forms double strands at certain regions with a single stranded loop. The 3′ end terminates with – CCA sequence and the 5′ end with G or C.
In addition to bases A, G, C and U present in tRNA, certain unusual bases are also found. These unusual bases are absent in other RNAs. The unusual bases are formed by specific chemical modification such as addition of methyl (-CH3) group to form 3-methyl cytosine or 1-methylguanosine, deamination of adenosine to inosine, reduction of uracil to dihydrouracil or rearrangement of uracil into pseudo uracil.
The other unusual bases are methyl guanine (Gme), dimethyl guanine (Gme2), methyl cytosine (Cme), ribothymine (T), pseudouridine (Ѱ), dihydrouridine (DHU, H2U, U2), inosine (I) and methylinosine (Ime).
Most of the bases pair according to Watson and Crick’s model but unusual bases do not because of bringing about changes due to substitution or alterations in those positions that take part in hydrogen bonding. The unusual bases protect the tRNA molecules from break down by RNAase. Consequently several non-base paired loops are formed in tRNA.
Clover Leaf Model of tRNA:
For the first time R. Holley (1968) prepared the clover leaf model for yeast tRNA alanine (tRNAala) which includes several known functions of tRNA. This model has been well accepted.
A typical clover leaf model is shown in Fig. 5.13 which reveals the following features:
(i) The single polynucleotide chain of all the tRNA molecule is folded upon itself to form five arms e.g. acceptor arm, DHU arm, anticodon arm, variable arm and T\|/C arm. An arm consists of a stem and a loop. Except the acceptor arm, the other arms consist of their respective stem and loop.
(ii) The acceptor stem consists of 7 base pairs and 4 unpaired bases, the unpaired bases contain a three -CCA bases and a forth variable purine (A or G) at 3′ end or polynucleotide chain. The last residue, adenylic acid (A) acts as amino acid attachment site. The 5′ end of tRNA contains either (G) or (C).
(iii) The DHU (dihydrouridine) loop constitutes 7-12 unpaired bases, and acts as the site for recognition of amino acid activating enzyme aminoacyl tRNA synthetase. It consists of a total of 15-18 nucleotides (3-4 base pairs and 7-11 unpaired bases) in the loop. It has two variable regions a and P on both sides of guanine residues. These two regions contain 1-3 nucleotides, often pyrimidines.
(iv) All the tRNA molecules contain different nucleotide triplet codons on anticodon loop. It is also called anticodon or codon recognition site. It is complementary to the corresponding triplet codon of the mRNA molecule.
The anticodon stem consists of 5 base pairs, and the anticodon loop contains 7 unpaired nucleotides. The middle three nucleotides act as anticodon which identify three complementary bases of mRNA molecule. There is a hyper modified purine (HPu) on 3′ side chain of anticodon.
(v) The tRNA also possesses a TѰC arm that consists of a stem of 5 base pairs and a loop of 7 unpaired bases including pseudouridine. The TΨC loop consists of a TΨC sequence at 5′ → 3′ direction. The TΨC arm has a ribosome recognition site and binds the tRNA molecules to the ribosome.
(vi) In some tRNAs with long chain, a variable arm of extra arm is present between the anticodon arm and TΨ arm. The variable arm may or may not contain a stem. The electron photomicrograph reveals a tertiary structure of tRNA where the different limbs are separately formed by the acceptor, TΨC and DHU arms and anticodon arm are visible (Fig. 5.14). These limbs formed by hydrogen bonds are found between bases and ribose-phosphate backbone, and between the residues of backbone.
The tRNA that initates protein synthesis is called initiator tRNA. The initator tRNA of eukaryotes differs from the prokaryotes. The tRNA specifies methionine as the starting amino acid in eukaryotic protein synthesis and N-formyl methionine in prokaryotes. Therefore, the two tRNAs specific to these two amino acids are methionyl tRNA (tRNA/-met). These two tRNAs differ from each other.