Let us make in-depth study of the chemical nature, types of ribonucleic acid (RNA) and also about RNA world.

Chemical Nature:

RNA is a single stranded mixed polymers of four types of ribotides linked together by 3′, 5′- phosphodiester bonds. They are adenylate (AMP), guanylate (GMP), cytidylate (CMP) and uridylate (UMP).

RNA is predominantly single stranded nucleic acid. However, it may fold back on itself to form an anti-parallel duplex structure called a hairpin which consists of a base-paired stem (like A-DNA) and a loop of unpaired bases.

RNA came first in evolution, having both genetic and catalytic properties. Later DNA replaced RNA as more stable molecule for genetic information and proteins carried out the catalytic role while RNA acts as the intermediate between the two.

Size:

RNA molecule is much smaller in size than DNA. It consists of up to 12,000 nucleotides whereas DNA consists of up to 4.3 million nucleotides.

Location:

RNA found in both prokaryotic and eukaryotic cells. In eukaryotic cell RNA found in cytoplasm as well as in nucleus. In the cytoplasm it occurs freely as well as in the ribosomes while in the nucleus it is present in association with chromosomes. RNA also found in matrix of mitochondria and stroma of chloroplast.

Types of RNA:

On the basis of function, RNA is of two types, viz.

(i) Genetic RNA and

(ii) Non-genetic RNA.

(i) Genetic or Genomic RNA:

It occurs in riboviruses and viroid’s. It is single stranded in TMV, HIV, Influenza viruses etc. while double stranded in Reovirus. In TMV genetic RNA is (+) RNA strand that directs the synthesis of a (-) RNA strand. Then the (-) RNA strand serves as the template for the synthesis of a large number of (+) RNA strands. In retroviruses like HIV Rous sarcoma viruses the genetic RNA is the (+) strand that directs the synthesis of DNA by reverse transcription.

(ii) Non-Genetic RNA:

Where DNA is the genetic material RNA is said to be non-genetic. Such RNA is synthesized from DNA template by the process called transcription. Transcription is catalyzed by RNA polymerase. In prokaryotes a single type of RNA polymerase can transcribe all types of RNA while in eukaryotes three different types of RNA polymerases (RNA Pol I, RNA Pol II and RNA Pol III) do the same job. The non-genetic RNA is mainly of 3 types – m RNA, tRNA and rRNA. The other types are hnRNA, snRNA,scRNA etc.

(a) Messenger RNA (mRNA):

The mRNA carries the coded information (genetic code) from DNA to ribosomes for synthesis of polypeptides. Hence, it is named messenger RNA. It constitutes about 5- 10% of total cellular RNA. It is most heterogeneous in size and stability.

The molecular weight of mRNA is about 500,000. Its sedimentation coefficient is 8S. In bacteria it is short lived. For example, in E. coli, the average half life of some mRNA is about 2 minutes. However, in mammals, it may live for many hours and even days. New mRNA is synthesized during early cleavage on a DNA strand in the presence of RNA polymerase enzyme.

Synthesis of mRNA differs from DNA replication in following three main aspects:

1. Ribose nucleotides are used instead of deoxyribose nucleotides.

2. Adenine pairs with uracil instead of thymine.

3. Only one strand of DNA leads to formation of mRNA molecule.

INFORMOSOME = mRNA+protein

The mRNA of prokaryotes differs from that of eukaryotes in several aspects.

Prokaryotic mRNA:

1. Majority is polycistronic, i.e., controlled by several cistrons.

2. It has short life, about two minutes.

3. Translation begins when mRNA is being synthesized on DNA template.

4. It undergoes little processing after synthesis.

Eukaryotic mRNA:

1. It is monocistronic, i.e., governed by one cistron.

2. It has long life, about few hours or days.

3. Translation begins only after transcription is over.

4. It undergoes considerable processing before formation of mRNA.

In eukaryotes all mRNAs are monocistronic i.e. each represents a single gene. But in prokaryotes, the majority is polycistronic mRNAs carrying coding sequences for many polypeptides while some mRNAs are monocistronic.

Each mRNA has two types of regions: The coding and non-coding regions. The coding region consists of a series of base triplets called codons starting with AUG (or GUG) and ending with a termination codon (UAA, UAG or UGA). In monocistronic mRNA, non-coding regions present at both ends. The 5′ non-coding region preceding the start codon is called a leader and the 3′ non-coding region following the termination codon is called a trailer.

In case of bacterial mRNA the leader contains a purine rich region called Shine-Dalgarno sequence present about 10 nucleotides upstream to start codon. This is recognized by 16S rRNA of smaller subunit of 70S ribosome and helps in initiation of protein synthesis. Similar sequence found in eukaryotic mRNAs that surround the AUG is called Kozak consensus sequences.

mRNA

In polycistronic mRNA, the coding regions are separated by intercistronic regions which may be 1-30 nucleotide long (even longer in phage RNAs). Rarely two coding regions overlap, so that the last base of the UGA termination codon of one coding region becomes the first base of the AUG, the start codon of the next coding region.

In eukaryotic mRNA, the 5′ end has a methylated cap (7 methylguanosine triphosphate or 7mGTP) while the 3′ end contain a stretch of20-250 adenine residues called poly(A) tail. The poly (A) tail is not coded in the DNA, but is added to the RNA in the nucleus after transcription.

In eukaryotes, RNA polymerase II (RNA Pol II) synthesizes the primary mRNA molecule called pre-mRNA or hnRNA (heterogeneous nuclear RNA) that localized in the nucleus. The pre-mRNA undergoes a process of maturation to become a mature mRNA which then enters into cytoplasm to serve as templates for protein synthesis. The maturation of pre-mRNA involves at least 3 steps — 5′ capping, polyadenylation and splicing.

(b) Transfer RNA or (tRNA):

The tRNA is also known as soluble RNA (sRNA) or adaptor RNA. It is the smallest known RNA species that constitute about 10-15 % of the total cellular RNAs. There are at least 20 types of tRNA molecules in every cell, one corresponding to each of the 20 amino acids required for protein synthesis. However, tRNA is always more than 20 and each amino acid is represented by more than one tRNA. Multiple tRNAs representing the same amino acid are called isoaccepting tRNAs. Although tRNAs are less stable in eukaryotes they are more stable in prokaryotes. The opposite is true for mRNAs.

Structure or tRNA (A) 2D- Clover leaf model (B) 3D-L Shaped structure

The primary structure of tRNA is 74 to 95 nucleotides long, but most commonly 6 residues. Their molecular weight is about 25,000 to 30,000. Except for the usual A, G, C and U, they contain many unusual bases such as pseudouridine (Ψ), dihydrouridine (D), inositol (I), ribothymidine (T), isopentenyladenosine (i6A), thiouridine (s4U) and methylguanosine (M1G). All these unusual bases are the modifications of one of the four bases created post-transcriptionally. The 5′ end of tRNA always ends in phosphorylated guanine (pG), whilse the 3′ end always ends in the – CCA sequence.

All tRNAs have a common secondary structure that appears like cloverleaf (R. W. Holley, 1965) due to base pairing between short complementary regions. The secondary cloverleaf model contains four major arms and one variable arm.

1. Acceptor arm composed about 7bp stem that ends in an unpaired sequence (5′ CCA 3′).

2. D arm consists of a stem (3 or 4 bp) and a loop called D-loop (DHU – loop) that contains the base dihydrouridine.

3. Anti-codon arm consists of a stem (5bp) and a 7 residue loop having anti-codon triplet complementary to the codon (a triplet of bases in mRNA).

4. TΨC arm(T arm) composed of a 5bp stem and a loop containing the triplet base sequence TΨC.

5. Extra arm (variable arm) lies between TΨC and anti-codon arms. On the basis of extra arm, tRNAs are of 2 types- Class 1 tRNAs have small extra arm (3-5 bp long) and constitute 75% of all tRNAs. Class 2 tRNAs have a large extra arm (13-21 bp) and often have a stem-loop structure.

The tRNA is functional in its tertiary structure and appears L-shaped (by Kim). It is formed by the folding of cloverleaf structure and is stabilized by nine hydrogen bonds (tertiary hydrogen bonds) occurs between residues of D-arm and TΨC arm.

The tRNA without amino acid is called uncharged tRNA. The tRNA attached to amino acid is called charged or aminoacyl tRNA. The charging of tRNA is catalyzed by enzyme aminoacyl -tRNA synthetase and the process is called amino-acylation. The tRNAs function as translational adaptors because at one hand they recognize specific codons of mRNA through anti-codons and on the other hand deliver amino acids to the ribosome.

(c) Small stable RNA (ssRNA):

They are discrete, highly conserved RNA molecules consist of 90- 300 nucleotides. They are of two types, small nuclear RNAs (snRNA) restricted to nucleus and small cytoplasmic RNAs (scRNA). Naturally they exist as ribonucleoprotein particles i.e. snRNP (snurps) and scRNP (scyrps)

(d) Ribosomal RNA (rRNA):

The RNA which is found in ribosomes is called ribosomal RNA. It is most abundant and constitutes about 80% of the total cellular RNA. The rRNA molecule is highly coiled. In combination with proteins, it forms small and large subunits of the ribosomes, hence its name.

The main features of rRNA are given below:

1. Ribosomal RNA is more stable than mRNA.

2. Ribosomal RNA is synthesized from nucleolar DNA in eukaryotes and forms a part of DNA in prokaryotes.

3. Synthesis of rRNA begins during gastrulation and increases as embryo develops.

4. On the basis of molecular weight and sedimentation rate, rRNA is of three types viz., over a million (21S-29S RNA), (2) with molecular weight below one million (12S-18S) and (3) with low molecular weight (SSRNA).

5. In addition to the formation of subunits of ribosomes in combination with proteins, its other function is binding of mRNA and tRNA to ribosomes.

6. The rRNA genes and their sequences are conserved through billion years of evolutionary divergences. Hence, by comparing the sequences of rRNA genes the possible phylogeny of organisms can be ascertained.Structure of rRNA

RNA World:

The phrase “RNA World” was first used by Nobel laureate Walter Gilbert in 1986 due to catalytic properties of various forms of RNA. The RNA World concept claims that RNA was the original genetic material in early organisms.

It is thought that RNA preceded DNA because:

(a) It is single stranded but DNA has two – so DNA possibly ‘evolved’ from RNA

(b) It has faster mutation rates during replication than DNA does

(c) It codes for proteins directly, unlike DNA – which is transcribed into mRNA, which is then translated to amino acids

(d) Some RNA also has enzymatic function – a feature that is thought to have been essential for primitive organisms

RNA as an Enzyme:

RNA enzymes or ribozymes, are found in today’s DNA-based life and could be examples of living fossils. Ribozymes play vital roles & helps to catalyze peptide bond synthesis during protein synthesis inside the smallest living most abundant cell organelle Ribosome.

RNA as a Regulator:

Riboswitches have been found to act as regulators of gene expression, particularly in bacteria, but also in plants and archaea. Riboswitches alter their secondary structure in response to the binding of a metabolite. This change in structure can result in the formation or disruption of a terminator, truncating or permitting transcription respectively. Alternatively, riboswitches may bind or occlude the Shine-dalgamo sequence, affecting translation. It has been suggested that these originated in an RNA-based world.

RNA in Information Storage:

RNA is a very similar molecule to DNA, and only has two chemical differences. The overall structure of RNA and DNA are immensely similar—one strand of DNA and one of RNA can bind to form a double helical structure. This makes the storage of information in RNA possible in a very similar way to the storage of information in DNA. However RNA is less stable.

Implications of the RNA World:

1. RNA world hypothesis, if true, has important implications for the definition of life. For most of the time that followed Watson and Crick’s elucidation of DNA structure in 1953, life was largely defined in terms of DNA and proteins: DNA and proteins seemed the dominant macromolecules in the living cell, with RNA only aiding in creating proteins from the DNA blueprint.

2. RNA world hypothesis places RNA at center-stage when life originated. This has been accompanied by many studies in the last ten years that demonstrate important aspects of RNA function not previously known-and supports the idea of a critical role for RNA in the mechanisms of life.

In 2001, the RNA world hypothesis received a boost with the deciphering of the 3-dimensional structure of the ribosome-which revealed that the key catalytic sites of ribosomes are composed of RNA, and that proteins hold no major structural role and are of peripheral functional importance.

Specifically, peptide bond formation, the reaction that binds amino acids together into proteins, is now known to be catalyzed by an adenine residue in the rRNA: the ribosome is a ribozyme. This finding suggests that RNA molecules were most likely capable of generating the first proteins.

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