Ribonucleic Acid (RNA) - Types

 

Most Prokaryotic and Eukaryotic cells contain another important nucleic acid the Ribonucleic Acid (RNA) besides DNA. Some viruses, however, contain no DNA but only RNA and in them RNA being the genetic molecule, carries the responsibilities of DNA. Such RNA is called genetic RNA. Further, in those cells in which the genetic substance is DNA, there occur another kind of RNA molecules which are called non- genetic RNA’s  and which have a DNA dependent synthesis (transcription).

Most cellular RNA is single stranded, although some viruses (e.g., Reovirus) have double stranded RNA. The single stranded RNA is folded upon itself either entirely or in certain regions. In the folded regions a majority of the bases are complementary and are joined by hydrogen bonds. This helps in the stability of the molecule.

Like DNA, RNA is polymeric nucleic acid of four monomeric ribotids or ribonucleotides. Each ribonucleotide contain a pentose sugar (D-ribose); a molecule of phosphate group and a nitrogen base. The nitrogen bases of RNA are two purines, adenine and guanine and two pyrimidines, cytosine and uracil. All normal RNA chains either start with adenine or guanine.

Three types of cellular RNA have been distinguished: messenger RNA (mRNA)or template RNA, ribosomal RNA (rRNA) and soluble (sRNA) or transfer RNA (tRNA). Ribosomal and transfer RNA comprise about 98% of all RNA.

Ribosomal RNA:

Ribosomal RNA (rRNA) constitutes about 80% of the total cellular RNA. It is found primarily in the ribosomes although, since it is synthesized in the nucleus, it is also detected in the nucleus.

Base sequence or rRNA is complementary to that of the region from where it is synthesized. In prokaryotes, the rRNA molecule is formed on the part of the DNA strand called the ribosomal DNA. In eukaryotes, ribosomes are formed on the nucleolus.

The nucleolar organizer contains ribosomal DNA which transcribes rRNA. Ribosomal RNA is formed from only a small section of DNA molecule, and hence there is no definite base relationship between rRNA and DNA as a whole.

The base ratios of rRNA ar very similar in ribosome from many different organisms. This suggests a general structural similarity. Depending upon ionic strength, temperature and pH the RNA molecule may be short compact rod, a compact coil or an extended strand. It consists of a single strand twisted upon itself in some regions. It has helical regions

 The ribosomes consist of proteins and RNA. The 70s ribosome of prokaryotes consist of 30s and 50s. The 30s subunit contains 16s RNA, while the 50s subunits contains 23s and 5s RNA.  The 80s Eukaryotic ribosome consists of 40s and 60s subunits.  In vertebrates the 40s subunit contains 16- 18s rRNA, while 60s subunit contains 28 - 29s, 5.8s and 5s rRNA. In plants and invertebrates the 40s subunits contents 16-18s rRNA, while 60s subunit contains 25s, 5s and 5.8s rRNA.

 Synthesis of rRNA

In prokaryotes genes the sequences specifying 16s, 23s and 5s are arranged in a series. mRNA is transcribed from DNA as a unit which has been called P30s. Experimental evidence indicates that the 30s unit has the 16s component at the 5’ prime and 23s component at the 3’prime and with spacer unit between the two components. In some prokaryotes 5s RNA is transcribed at or near the 3’end

 During processing the P30s transcriptional unit is cleaved within the spacer segment by RNase III into 25s and 18s segments. These are then reduced to P23s and p16s segments respectively, also by RNase III. Secondary trimming of these intermediates is the final size, 23s and 16s respectively. The enzyme involved in secondary trimming is probably a ribonuclease, designated as RNase M or mutase.

 Messenger RNA

Jakob and Monad (1961) proposed the name messenger RNA for the RNA carrying information for protein synthesis from DNA to the site of protein synthesis (ribosomes). It consists of only three to five percentage of the total cellular RNA.

The molecular weight of an average size mRNA molecule is about 500,00 Dalton. It is always single stranded.  It contains mostly the base adenine, guanine, cytosine and uracil. There are few unusual substituted bases. There is no base pairing in mRNA. In fact, base pairing in the mRNA strand destroys its biological activity.

mRNA synthesis in Prokaryotes

Synthesis of mRNA is accomplished with only one of the two strands of DNA which is used as template. The enzyme RNA Polymerase joins the ribonucleotides, thus catalyzing the formation of 3’ to 5’ phosphodiester bonds that form the RNA backbone. In this synthesis the AU/GC ratio of RNA is similar to the AT/GC ratio of DNA. The synthesis is initiated at 5’ end and the direction of growth is 5’ to 3’.

In bacteria, the process of transcription of mRNA  is simultaneous with translation i.e., as soon as the mRNA is being transcribed by RNA polymerase the ribosomes become attached to the mRNA to initiate protein synthesis.

mRNA synthesis in Eukaryotes

The origin and fate of mRNA in Eukaryotic cell is much more Complex than in prokaryotes it consists of a complex series of steps that i. comprise the actual transcription of DNA into mRNA precursors, ii.  the intranuclear processing or tailoring of these precursors and iii. the transport of mRNAs into cytoplasm and their association with ribosomes to initiate the process of translation.

Consequently, synthesis of mRNA molecules includes following events:

1. Heterogenous nuclear RNA:

mRNA is synthesized in the nucleus as the part of a heterogenous population of large RNA molecules which constitute the so called heterogeneous nuclear RNA ( het-RNA or HnRNA).

The HnRNA is also called high molecular weight RNA or DNA-like RNA (dRNA). The molecules of HnRNA range in size from 5×10⁵ to 10⁷  daltons and are degraded for the most part within the nucleus at a relatively rapid rate. Only about 20 percent of the HnRNA in terms of total nucleotide, is not degraded and convert into mRNA molecules.

Before leaving the nucleus, Eukaryotic mRNA undergo three kinds of modifications:

 

i.  the addition of polyadenylate (poly A) at the 3’ end by Poly(A) polymerase to prevent them from getting digested by Nucleases

ii.   the addition of ‘caps’ of special nucleotides at the 5’end by guanyl transferase enzymes which adds GTP to its 5’end and methyl transferase perform methylation by transferring methyl group to the Guanine caps.

 Besides, these post-transcriptional modifications, eukaryotic mRNA’s differ from prokaryotic mRNAs in leaving the nucleus prior to their translation.

 

Heterogeneity and types of mRNA:

When the total mRNA population of an organism is considered, it is found to be heterogenous in size, showing a wide range of S values of 6 to 30. This property of mRNA reflects the fact that the size or length of mRNA molecule is directly related with the size or length of the codons for different protein molecules. According to size, following two types of mRNA molecules can be recognized:

a.     Monocistronic mRNA:

Mostly the mRNA carriers the codons for single cistron, i.e., codes for one complete protein molecule of DNA. Such mRNA molecule is called monocistronic mRNA.

b.     Polygenic or Polycistronic mRNA:

mRNA molecule carries codes for several adjacent DNA cistrons i.e., it carries codes for more than one protein molecule. It contains several sites for initiating and terminating polypeptide synthesis. This type of mRNA is called polygenic or polycistronic mRNA.

 

Life Span of mRNA:

            In most prokaryotic and eukaryotic cells, mRNA has short lifetime. For example, average life of mRNA of E.coli is about 2 minutes because it is attacked by the cytoplasmic ribonuclease enzyme. So that, most times, mRNA makes up only 5% of the total cellular RNA.

Likewise, in most eukaryotes, the average life span of mRNA si one to four hours, however, in both  bacteria and eukaryotes mRNA are known that are metabolically stable and apparently resistant to nucleases.

For example, unmature red blood cells (reticulocytes) of mammals mRNA exists upto 2 days for prolong utilization in the synthesis of globin protein of heamoglobin.

 

Transfer RNA (tRNA)

The RNA which possess the capacity to combine specifically with only one amino acid in a reaction mediated by a set of amino acid specific enzymes called aminoacyl -tRNa synthetases; transfers the amino acid from the ‘amino acid pool’ to the site of protein synthesis and recognizes the codons of the mRNA is known as the soluble RNA (S RNA) or transfer RNA (tRNA).

Thus, tRNA molecule acts as an interpreter of genetic code and has to perform several highly complex functions during protein synthesis – it interacts with a specific synthetase enzyme, possesses a site for binding of amino acid, possesses a second site for interacting with a ribosome, and contains an anticodon that must be expected to the codons of mRNA.

Structure and Maturation of tRNA:

The tRNA molecules that perform all these functions account to 10-20%  of total RNA of the cell. Each tRNA has a sedimentation coefficient of 3.8s and contains 75 to 80 nucleotides. The sequence of Alanine tRNA was first of all elucidated by R.W Holley and collaborators 1965. The primary structure of some 45 tRNA, form E.coli to mammalian liver cells to higher plant cells, is already known.

Nuclear DNA transcribes precursor tRNA through RNA polymerase. Precursor tRNA consists of 120-130 bases. Like rRNA and mRNA molecules, molecules of tRNA are formed to be matured or tailored in the nucleus prior to their movement to the cytoplasm.

For example, in E.coli the precursor of tRNA molecules have been isolated, each of which has about 40 extra nucleotides, principally at 5’end but also at the 3’end. These extra nucleotides are subsequently cleaved off probably by RNase P and RNase Q or RNase pIII, to yield a molecule of the final 70 to 80 nucleotides. The bases 5’-CCA-3’ are added to the 3’end of every tRNA molecules regardless of its amino acid affinity, by an enzyme called tRNA phosphorylase.

Several model for the secondary structure of tRNA have been proposed, and of these the Clover leaf model of Holley is the most widely accepted.

According to these model, the single polynucleotide chain of tRNA is folded upon itself to form 5 arms. As a result of the folding 3’ and the 5’ ends of the chain come near to each other. An arm consists of a stem and loop. In the double helical stems there is internal Watson-Crick base pairing which follows the A-U and G-C combinations. There is no base pairing in the loops.

All tRNA molecules contain the same terminal sequence of 5’-CCA-3’ bases at 3’-end of the polynucleotide chain. The last residue, adenylic acid (A),is the amino acid attachment site. The amino acid is attached to the second or third carbon of the ribose sugar of the terminal nucleotide.

The second arm is called the D arm. It consists of the of 15-18 nucleotides in the loop. The loop of the D arm is called Loop I or dihydrouracil (DHU) loop, named for the modified uracil basses this region always contains. It contains site for recognition of the amino acid activating synthetase enzyme.

The third arm is called the Anticodon arm. In this arm the stem has 5 paired baes and the loop has 7 unpaired bases. The loop is called the anticodon loop. Three of the 7 unpaired bases in the loop determine the pairing of tRNA with the specific codon of mRNA.

The fourth arm is called TΨC arm. Its loop has ribosome recognition site.

The variable arm has a loop with 4-5 bases. The stem may or may not be formed.

 

Functions:

The tRNA picks up a specific activated amino acid from the amino acid pool in the cytoplasm. The amino acid is transferred to the ribosome in the cytoplasm where protein synthesis takes place. .