In this article we will discuss about RNA (Ribonucleic Acid). After reading this article you will learn about: 1. Structure of RNA 2. Types of RNA.
Structure of RNA:
The primary structure of RNA is the same as that of DNA. It is also a polynucleotide chain with 5′-3′ sugar phosphate links. But the sugar is ribose and generally it exists as a single-stranded molecule. For that reason, it does not have the one-to-one ratio between the complementary bases. The amount of purines is not equal to that of pyrimidines.
One of the four major bases in RNA is uracil (U) instead of thymine. Biophysical properties of RNA show much less secondary structure. However, when a sequence of bases is followed by a complementary sequence in the same chain, the polynucleotide may fold back on itself to generate an antiparallel duplex structure, known as a hairpin.
It has a stem, the base-paired double helical region, and a loop with unpaired bases at one end (Fig. 3.55). In these regions G can also pair with U, but it is not as strong as G-C pair.
Cells contain three types of RNA-messenger RNA (mRNA), ribosomal RNA(rRNA) and transfer RNA (tRNA). Messenger RNA serves as the template for protein synthesis. It is a very heterogeneous class of molecules and very unstable. It constitutes 2 – 5 per cent of the total RNA of the normal cell. It was first detected by Hershey (1956). The name and concept of messenger RNA was first given by F. Jacob and J. Monod (1961).
When molten DNA is slowly cooled with some specific m-RNA, DNA- RNA hybrid molecules are formed, suggesting that m-RNA is formed from the template strand of the DNA duplex.
It is very heterogeneous in size because genes or groups of genes vary in length. In prokaryotes, the translation of mRNA molecule very often begins before the completion of its transcription, because mRNAs are both transcribed as well as translated in the 5′ to 3′ direction and both the processes are not separated by nuclear membrane.
(i) The Structure of mRNAs:
Messenger RNAs encode the amino acid sequences of proteins. The number and types of distinct mRNA species (transcriptomics = mRNA profile) in a given cell at a particular phase of life of an individual is comparable to the number of different proteins (proteomics = protein profile) present in the cell at that phase. Messenger RNAs constitute 1 % to 2% of total cellular RNA.
All mRNAs share certain properties:
(a) They contain a continuous sequence of nucleotides encoding a specific polypeptide;
(b) They are found in the cytoplasm; and
(c) They are either attached to ribosomes or capable of such an attachment in order to be translated.
(d) Consistent with the range of molecular masses observed for polypeptides, mRNAs vary in length from a few hundred nucleotides to several thousand nucleotides;
(e) mRNAs generally are more labile than rRNAs and tRNAs, although the turnover rate for some mRNAs is quite slow.
Most mRNAs contain a significant noncoding segment, that is, a portion that does not direct the assembly of amino acids. For example, approximately 25 percent of each globin mRNA consists of noncoding, untranslated regions (UTRs). Noncoding portions are found on both the 5′ and 3′ ends of a messenger RNA and contain sequences that have important regulatory roles.
In addition to noncoding nucleotides, eukaryotic mRNAs have special modifications at their 5′ and 3′ termini that are not found on prokaryotic messages. The 5′ end of eukaryotic mRNAs has a methylatedguanosine “cap,” while the 3′ end has a string of 50 to 250 adenosine residues that form a poly(A) tail.
The sequence between the initiation and termination codons constitutes the open reading frame (ORF). It is flanked by 5′ and 3′ untranslated regions that are highly variable in length. Consequently, the length of mRNA does not always correlate with the length of the amino acid sequence it encodes.
The precise function of the 5′ and 3′ UTRs is still poorly understood for many mRNAs, but often these regions contain RNA sequences that can form secondary structures and interact with proteins, which regulate transport, translation and stability of the mRNA.
The 3′ polyadenylate tail is not encoded in the DNA, but is added post-transcriptionally before the mRNA is exported from the nucleus to the cytoplasm. An enzyme complex containing an endonuclease and poly(A) polymerase recognizes a signal sequence (5′ – AAUAAA – 3′) near the 3′ end of the transcript, cleaves the pre-mRNA downstream from the signal, and adds 20 to 250 adenosine residues to the 3’end.
The structure of plastid and mitochondrial mRNAs differs from that of cytoplasmic mRNA. The organellar mRNAs fack both the 5′ cap and poly(A) tail. As a result of differences in post transcriptional RNA processing, mitochondrial mRNAs commonly retain 5′ triphosphate group, while the 5′ ends of most plastid mRNAs are monophosphorylated.
Similar to prokaryotic mRNAs, the 5′- and 3′-UTRs of most organellar mRNAs can form stem-loop structure that have regulatory and stabilizing functions. These RNA structures often interact with proteins and serve as signals that affect the processing, translation, and degradation of the mRNA.
Eukaryotic cells contain additional classes of small, stable RNAs. Stable uridine-rich RNAs are components of small nuclear ribonucleo-proteins (UsnRNPs) and make up a major class of the small nuclear RNAs (snRNAs) of eukaryotes. These RNAs are numbered from U1 onward. Some snRNAs occur in high copy number in the nucleoplasm and are known as major snRNAs.
Most of the minor snRNAs are found in the nucleolus and are therefore designated snoRNAs (small nucleolar RNAs). Most of the snRNAs in snRNPs are involved in the splicing of intervening non-coding regions from the precursor mRNAs in the nucleus.
Other snRNAs participate in processing of the 3′ terminus of histone pre-mRNA, which lacks a poly (A) tail. Most of the snoRNAs are involved in the processing of rRNA precursors.
In eukaryotes transcription and translation are not simultaneous, as transcription is a nuclear process and translation is a cytoplasmic process. The newly transcribed mRNAs are called primary gene transcripts, which are transported out of the nucleus to the cytoplasm.
The functional mRNAs of eukaryotes are derived from the primary gene transcripts through several types of processing as follows:
(a) Cleavage of pre-mRNA or large mRNA precursor to small individual mRNA molecules.
(b) Capping or addition of 7-methyl guanosine groups to the 5′ ends of the molecule.
(c) Poly (A) tailing or the addition of 200 nucleotide long sequence of adenylate nucleotide to the 3′ end of the mRNA molecule.
(d) Formation of complexes with specific proteins.
The base sequence from 5′ end to the initiation codon, is called leader sequence and the intervening non-coding sequences are called introns. The coding sequences, which are translated into protein are called exons. The processing of the primary transcript involves the removal of the intron sequences.
The post transcriptional modifications of individual gene transcripts may undergo some or all of the above mentioned 4 types of processing.
Most of the non-ribosomal RNAs synthesized in the nuclei of eukaryotic cells are very large and highly variable in size and are called heterogenous nuclear RNA(hnRNA) or pre-mRNA.
Rapid processing of these giant molecules inside the nucleus soon after their transcription, results in the formation of mRNA molecules that are transported out of the nucleus to the cytoplasm. During processing, from the primary transcripts the introns are spliced out.
There are three distinct types of splicing mechanisms as follows:
(a) Splicing of tRNA precursor by endonuclease and ligase enzyme.
(b) Autosplicing of Tetrahymena RNA precursor is a unique reaction mechanism, catalyzed by the RNA molecule itself (self-splicing). These catalyting RNA molecules are called ribozymes. In such type of splicing mechanism no protein enzyme is involved.
This autocatalytic activity has also been shown to occur in rRNA precursors of several lower eukaryotes and in a large number of mRNA, tRNA and mRNA precursors in mitochondria and chloroplast of many different species.
(c) The introns of pre-mRNA transcripts are spliced out in two-step reactions catalyzed by ribonucleoprotein complexes called spliciosomes.
The introns of plant nuclear pre-mRNAs vary, tend to be AU-rich and have conserved sequences at their splice junctions. In dicots, these introns contain 70% AU, whereas the same in monocots is 60%. The nucleotide sequences surrounding each exon-intron junction are highly conserved.
In almost all of the nuclear mRNA introns of plants, the boundary sequences at the 5′ splice site (donor site) and the 3’splice site (acceptor site) consist of 5′ GU and AG 3′. This characteristic is known as the GU-AG rule. An additional structural element, the branch site, is usually located 20 to 40 nucleotides upstream from the 3’splice site.
During intron excision, the 5’end of the intron binds to an adenine residue at the branch site, forming a lariat. Pre-mRNA intron splicing occurs within a large ribonucleoprotein complex(spliceosome), which contains the snRNAs U1, U2, the U4/U6 complex, and 115, which remain associated with small ribonuclear proteins (snRNPs), and non-snRNP protein factors (Fig. 3.58).
Four major types of introns are given in the table below:
Self-splicing mechanism varies depending on the group of introns. Splicing of group-l introns is initiated when a guanosine molecule or 5′ phosphorylated derivative binds to an intron sequence. The 3’OH group of the bound guanosine attacks the phosphate group at the 5’splice site, cleaving the phosphodiester bond at that site releasing the exon-1.
The 3′ OH at the end of the exon-1 reacts with the phosphate at the 3’splice site, ligating the two exons and releasing a linear intron (Fig. 3.59). The self-splicing mechanism of group-II introns resembles splicing of nuclear pre-mRNAs, but does not involve the formation of a spliceosome. These splicing reactions are typically intramolecular and join exons from a single transcript and is called cis-splicing.
In contrast, certain Group-II introns of plants are removed by a novel trans-splicing mechanism involving two or more RNA molecules. It is found in plant chloroplast and mitochondria. Only certain species of small, stable RNAs that are transcribed by RNA polymerase III do not undergo extensive processing.
Types of RNA:
(i) Ribosomal RNA:
The bulk of cellular RNA is ribosomal RNA. It is the principal component of ribosomes, but its definite role in protein synthesis is not clear. Plants, algae, and photosynthetic protists each contain three classes of ribosomes: cytoplasmic, plastid, and mitochondrial. In E. coli ribosomes three types of rRNA are found. They are called 5S, 16S and 23S due to their sedimentation behaviour.
Each ribosome contains one molecule of each of these rRNA species. Eukaryotic ribosomes, however, contain one molecule each of the 5S, 7S, 18S, and 28S rRNA species. Ribosomal RNA is the most abundant of the 3 types of RNA in the cell.
Ribosomal RNA is single stranded, but it also shows high degree of secondary modifications due to the formation of double strands between complementary regions and hair pin loops. Each loop contains duplex stems. The loops and the stems provide the specific vectorial binding sites for various ribosomal proteins and other enzymes for protein synthesis.
The 3′ end of 16S rRNA isolated from E. coli has a Shine and Dalgarno sequence which serves as mRNA binding site in the 30S ribosome. This sequence helps to recognize the starting end of the mRNA. The 5S rRNA has a sequence which is complementary to TΨC sequence of all tRNAs. So, 5S RNA is essential for the binding of tRNA to the ribosomes.
The processing of pre-rRNAs includes nucleolytic cleavage and methylation. Some eukaryotic rRNA transcripts undergo splicing by the intron itself (self-splicing, ribozyme).
The transcription units in the nuclear rRNA gene cluster that encode the 17S, 5.8S, and 25S rRNA molecules are transcribed by RNA polymerase I into a single long precusor molecule, which then undergoes a series of cleavages and methylation steps to yield 17S, 5.8S, and 25S rRNA molecules.
Transcription of the 5S rRNA from the corresponding nuclear genes is independent and is catalyzed by RNA polymerase III. It requires no processing.
The 17S, 5.8S, and 25S rRNAs are produced from a common 45S rRNA precursor molecule by processing reactions catalyzed by several RNases. During processing, the 25S and 5.8S rRNAs become hydrogen bonded and remain paired after the processing is complete (Fig. 3.60).
Four plastid rRNA molecules are encoded as a polycistronic transcription unit that also includes the two tRNA genes encoding tRNAala and tRNAlle in the spacer region that separate the 16S and 23S rRNA, each of which contains a long intron. Through a complex processing pathway the precursor RNA is cleaved into 16S, 23S, 4.5S, and 5S rRNAs and the tRNA precursors.
The cleaved tRNA precursors are then processed into functional tRNAlle and tRNAala molecules. The tRNAs are transcribed by RNA polymerase III from clusters of nuclear genes, but unlike 5S rRNA they need processing (Fig. 3.61).
(ii) Transfer RNA:
Transfer RNA was first identified as a fraction of RNA sedimenting at 4S. The tRNAs are 75-85 nucleotides long . They are also known as soluble RNA or cytoplasmic RNA. Transfer RNA is an adaptor molecule performing double functions. It recognizes both codon and the amino acid.
The nucleotide sequence of tRNA gives the form of a clover leaf. In this structure the complementary paired bases form stems for single stranded loops. The stem and loop structures are known as arms of tRNA.
The base sequence of a tRNA molecule was first determined by Robert Holley in 1965. Except for the usual A, G, C and U, they contain a variety of unusual nucleosides, such as pseudouridine (Ψ), dihydrouridine (H2U), ribothymidine (rT), inosine (I), mono- and dimethyl derivatives of adenosine and guanosine and their thiolated derivatives.
These modifications take place on one of the four bases only after it has been incorporated into the polyribonucleotide chain. There are four loops and four major arms in the tRNA molecule. The arms are named for their structures and functions.
The acceptor arm consisting of a base paired stem that always ends in an unpaired —C—C— A sequence whose free 3′ OH group is aminoacylated and the 5′ end generally terminates either in G or C.
The other arms consists of paired stems and unpaired loops. The D-arm is named for the presence of dihydrouracil base. The anticodon arm always contains the anticodon triplet in the centre of the loop. The TΨC arm is named for the presence of this triplet sequence.
The variable feature of the tRNA is the so called extra arm, lying in between TΨC and anticodon arms and on the basis of its nature tRNA are classified into two types – class-I tRNAs (having a small extra arm) and the class-II tRNAs (having a large extra arm). In clockwise direction around the clover leaf there are always 7 base pairs in the acceptor stem, 5 in TΨC arm, 5 in anticodn arm and usually-three in the D arm.
The position of nuclear tRNA introns is conserved, but their sequences are not. An endonuclease cleaves the pre- tRNA at both ends of the intron resulting in the formation of a cyclic 2′, 3′-phosphate group at the 3′ end of the 5′ tRNA segment, and a free 5′-hydroxyl group of the 3′ tRNA segment.
The cyclic phosphate group is cleaved to form a 2′-phosphate group. The free 5′ hydroxyl group is then phosphorylated and both the halves are joined by an RNA ligase. A 2′-phosphatase removes the 2′-phosphate group to yield the mature spliced tRNA.