Molecular biology WID Masters of Science in Tropical and Infectious Diseases-Transcription Lecture Series RNA I. Introduction and Background:

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1 Molecular biology WID Masters of Science in Tropical and Infectious Diseases-Transcription Lecture Series RNA I. Introduction and Background: DNA and RNA each consists of only four different nucleotides. All nucleotides have a common structure: a phosphate group linked by a phosphoester bond to a pentose (a fivecarbon sugar molecule) that in turn is linked to an organic base (Fig.1a) In RNA, the pentose is ribose; in DNA, it is deoxyribose (Fig. 1b). The only other difference in the nucleotides of DNA and RNA is that one of the four organic bases differs between the two polymers. The bases adenine, guanine, and cytosine are found in both DNA and RNA; thymine is found only in DNA, and uracil is found only in RNA. The bases are abbreviated as single letter codes: A, G, C, T, and U, respectively (Fig. 2). For convenience the single letters are also used when long sequences of nucleotides are written out. Fig. 2, The chemical structures of the principal bases in nucleic acids. In nucleic acids and nucleotides, nitrogen 9 of purines and nitrogen 1 of pyrimidines are bonded to the 1 carbon of ribose or deoxyribose. The base components of nucleic acids are heterocyclic compounds with the rings containing nitrogen and carbon. Adenine and guanine are purines, which contain a pair of fused rings; cytosine, thymine, and uracil are pyrimidines, which contain a single ring. The acidic character of nucleotides is due to the presence of phosphate, which dissociates at the ph found inside cells, freeing hydrogen ions and leaving the phosphate negatively charged. These charges attract proteins, so nucleic acids in cells are associated with proteins. University of Nairobi MSc Mol. Biol. Lecture 1 and 2: Introduction and RNA 1

2 RNA is a nucleic acid similar to DNA; RNA is a single stranded polynucleotide, because in RNA molecular structure the constituent nucleotides incorporate a ribose carbohydrate which is different from DNA, in which nucleotides incorporate a deoxyribose carbohydrate (Fig.1). There are four nucleotides in RNA, which are differentiated according to which base is bound to the carbohydrate in each - Adenine (A), Cytosine (C), Guanine (G) or Uracil (U). The third constituent part of nucleotides - a phosphate group - allows them to bind together into the polynucleotide called RNA (Fig. 3) through linkages such as the uracil adenine base pair (Fig.4). Fig. 3: The DNA and RNA polynucleotide chains have similar structures except for the presencee of uridine in RNA (instead of thymidine) and for the presence of the 2' OH in the ribose sugar. This 2' OH makes the RNA 3', 5 phosphodiester bond susceptible to hydrolysis. (Note: RNA - extra OH at 2 of pentose sugar) 1 Fig. 4 uracil-adenine base pair University of Nairobi MSc Mol. Biol. Lecture 1 and 2: Introduction and RNA 2

3 III. Types of RNA: There are three main types of RNA: Containing ribose, uracil and are usually singlestranded. i. Messenger RNA (mrna) is the RNA produced by the transcription of DNA strands by DNA polymerase. Genetic information is copied into mrna in the form of a commaless, overlapping, triplet (3 letter) code. Therefore mrna is the carrier of genetic information copied from DNA in the form of a series of three-base code words, each of which specifies a particular amino acid. mrna is then translated into proteins by ribosomes. (Fig.5) Fig. 5. The three roles of RNA in protein synthesis: Messenger RNA (mrna) is translated into protein by the joint action of transfer RNA (trna) and the ribosome, which is composed of numerous proteins and two major ribosomal RNA (rrna) molecules (Source: Molecular Biology of the Cell: Chapter 4). ii. Ribosomal RNA (rrna) is the type RNA that combines with different proteins to form ribosomes These complex structures, which physically move along an mrna molecule, catalyze the assembly of amino acids into protein chains. They also bind trnas and various accessory molecules necessary for protein synthesis. Ribosomes are composed of a large and small subunit, each of which contains its own rrna molecule (s) iii. Transfer RNA (trna) are short RNA sequences which function as intermediaries or adapters in ribosomal mrna to protein translation. Each type of amino acid has its own type of trna, which binds it and carries it to the growing end of a polypeptide chain if the next code word on mrna calls for it. The correct trna with its attached amino acid is selected at each step because each specific trna molecule contains a three-base sequence that can base-pair with its complementary code word in the mrna. Recently, other types of RNA have been described and are known as Non-coding RNA (ncrna) sequences: RNA sequences that function without being translated into a protein. Wide variety of ncrna are known and there are probably many that have yet to be discovered. Some examples: mirna (micro RNA): is a kind of helper RNA in mrna translation. (Geneticists are just learning about the functions of specific micrornas). Riboswitches Cis-acting regulatory sequences that respond to the environment RNase P RNA Component of RNase P, which edits trna. snorna (small nucleolar RNA) Direct rrna modification. University of Nairobi MSc Mol. Biol. Lecture 1 and 2: Introduction and RNA 3

4 SRP RNA (signal recognition molecule) Involved in the transport of secreted proteins to the endoplasmic reticulum. Telomerase RNA Structured RNA that provides sequence template for telomere sequences. tmrna (trna- mrna-like) Rescues stalled ribosomes and tags the protein product for degradation. III. RNA STRUCTURE CONFORMATION: Nucleoside triphosphates are used in the synthesis of nucleic acids (as well as many other functions in the cell): ATP is an energy carrier in the cell and GTP plays crucial roles in intracellular signaling and acts as an energy reservoir, particularly in protein synthesis. When nucleotides polymerize to form nucleic acids, the hydroxyl group attached to the 3 carbon of a sugar of one nucleotide forms an ester bond to the phosphate of another nucleotide, eliminating a molecule of water: The condensation reaction to form nucleic acid strands is similar to that in which a peptide bond is formed between two amino acids. (Fig. 6) Thus a single nucleic acid strand is a phosphate-pentose polymer (a polyester) with purine and pyrimidine bases as side groups. The links between the nucleotides are called phosphodiester bonds. Like a polypeptide, a nucleic acid strand has an end-to-end chemical orientation: the 5 end has a free hydroxyl or phosphate group on the 5 carbon of its terminal sugar; the 3 end has a free hydroxyl group on the 3 carbon of its terminal sugar. Fig. 6 This directionality, and that the fact that synthesis proceeds 5 to 3, has given rise to the convention that polynucleotide sequences are written and read in the 5 3 direction (from left to right); for example, the sequence AUG is assumed to be (5 )AUG(3 ). (Note: the letters A, G, C, T, and U stand for bases, they are also often used in diagrams to represent the whole nucleotides containing these bases.) The 5 3 directionality of a nucleic acid strand is an extremely important property of the molecule. University of Nairobi MSc Mol. Biol. Lecture 1 and 2: Introduction and RNA 4

5 The linear sequence of nucleotides linked by phosphodiester bonds constitutes the primary structure of nucleic acids. Polynucleotides can twist and fold into three-dimensional conformations stabilized by noncovalent bonds; in this respect, they are similar to polypeptides. Although the primary structures of DNA and RNA are generally similar, their conformations are quite different. Unlike RNA, which commonly exists as a single polynucleotide chain, or strand, DNA contains two intertwined polynucleotide strands. This structural difference is critical to the different functions of the two types of nucleic acids. RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base-pairs with complementary sequences found elsewhere on the same molecule. These interactions, along with additional nonconventional base-pair interactions, allow an RNA molecule to fold into a three-dimensional structure that is determined by its sequence of nucleotides. Fig. 7 (A) Diagram of a folded RNA structure showing only conventional base-pair interactions; (B) structure with both conventional ( ) and nonconventional ( ) base-pair interactions; (C) structure of an actual RNA, a portion of a group 1 intron. Each conventional base-pair interaction is indicated by a rung in the double helix. Bases in other configurations are indicated by broken rungs. In order for RNAs to carry out their function they have to adopt a secondary or tertiatiary structure: A secondary structure also provides insight into how a particular RNA functions. A secondary structure is needed for determining a tertiary structure. The primary structure of RNA is generally similar to that of DNA; however, the sugar component (ribose) of RNA has an additional hydroxyl group at the 2 position (see highlighted OH and H in Fig. 8), RNA is a long polynucleotide that can be double-stranded or single-stranded, linear or circular. Fig. 8 The various types of RNA exhibit different conformations. Differences in the sizes and conformations of the various types of RNA permit them to carry out specific functions in a cell. The simplest secondary structures in single-stranded RNAs are formed by pairing of complementary bases. Hairpins are formed by pairing of bases within 5 10 nucleotides of each other, and stem-loops by pairing of bases that are separated by 50 to several hundred nucleotides (Fig.9). The stem loops Stems are double-stranded regions of RNA that are A-form helices. They follow Watson-Crick base pairing rules. University of Nairobi MSc Mol. Biol. Lecture 1 and 2: Introduction and RNA 5

6 Fig.9 These simple folds can cooperate to form more complicated tertiary structures, one of which is termed a pseudoknot RNA secondary and tertiary structures:(a) Stem-loops, hairpins, and other secondary structures can form by base pairing between distant complementary segments of an RNA molecule. In stem-loops, the single-stranded loop between the base-paired helical stem may be hundreds or even thousands of nucleotides long, whereas in hairpins, the short turn may contain as few as 6 8 nucleotides. (b) Interactions between the flexible loops may result in further folding to form tertiary structures such as the pseudoknot. This tertiary structure resembles a figure-eight knot, but the free ends do not pass through the loops, so no knot is actually formed. Transfer RNA ( trna) The trna molecules are key to the translation process of the mrna sequence into the amino acid sequence of proteins (at least one type of trna for every amino acid). To be precise, the amino-acyl-trna-synthase proteins are the 'true' translators of the genetic code into an amino acid sequence. These synthetases acetylate trna molecules with the proper amino acid that corresponds to the anti-codon in the structure of the trna molecule. The anti-codon later recognizes the codon, the triple base sequence which 'codes' for the amino acid along the mrna strand. A failure of properly acetylating the trna with the right amino acid results in a amino acid mutation even though the DNA sequence has not been changed. trna molecules are small nucleic acids of nucleotides, mostly 76, with a molecular weight 18-20kD, with the secondary structure resembling a clover leaf. Here are a few common features shared by all trna molecules found in various organisms. (1) 5' terminus always phosphorylated (2) 7 bp stem, may have non-watson&crick pairing (like GU) acceptor or amino acid stem at 3' terminus, last three nucleotides CCA-3'-OH, amino acylation occurs at 3'-OH group of (3) 3-4 bp stem and loop contains the base dihydrouridine (D) [D- arm] (4) 5 bp stem and loop containing anti-codon triplet [anti-codon arm] (5) 5 bp stem and loop contains sequence TY C, Y standing for [T- arm] pseudouridine (6) variable arm (between anti-codon and T arm) of length 3-21 nucleotides (7) contains numerous modified bases (up to 25%) which are all post-transcriptionally modified University of Nairobi MSc Mol. Biol. Lecture 1 and 2: Introduction and RNA 6

7 The threee dimensional structure of trna resembles an L-shaped molecule (see below Fig.10) with the D-arm and anti-codon loop building one stretch and the T-arm and acceptor stem building the other stretch. The moleculee is about 6 nm in each direction with the antiare about 2. 0 codon to acceptor 3'-term ends being 7.6 nm apart. The diameter of both arms to 2.5 nm. Fig.10 trna molecules adopt a well-defined three-dimensional architecturee in solution that is crucial in protein synthesis. Larger rrna molecules also have locally well defined three-dimensional structures, with more flexible links in between. Secondary and tertiary structuress also have been recognized in mrna, particularly near the ends of molecules. These recently discovered structuress are under active study. Clearly, then, RNA molecules are like proteins in that they have structured domains connected by less structured, flexible stretches. The folded domains of RNA molecules not only are structurally analogous to theα helices and β strands found in proteins, but in some cases also have catalytic capacities. Such catalytic RNAs, called ribozymes, can cut RNA chains. Some RNA domains also can catalyze RNA splicing, a remarkable processs in which an internal RNA sequence, an intron, is cut and removed and the two resulting chains, the exons, are sealed together. This process occurs during formation of the majority of functional mrna molecules in eukaryotic cells, and also occurs in bacteriaa and archaea. Remarkably, some RNAs carry out self- splicing, with the catalytic activity residing in the intron sequence. University of Nairobi MSc Mol. Biol. Lecture 1 and 2: Introduction and RNA 7