RNA

RNA is a long un-branched macromolecule consisting of nucleotides joined by Phosphodiester Bridge. The sugar unit is ribose. The bases are Adenine, Uracil, and Guanine & Cytosine.

RNA molecules are usually single stranded, except some viruses. In fact the proportion of adenine differs from that of uracil and the proportion of guanine differs from that of cytosine, in most RNA molecules.

RNA molecules have region of double helical structure that are produced by the formation of hairpin loops. In these region A pairs with U and G pairs with C.

Cell contains several kinds of RNA:

1. Messenger RNA(mRNA):i) It is the template for protein synthesis. Also called the information carrier

in protein synthesis.ii) The base composition of the messenger should reflect the base

composition of the DNA that specifies it.iii) An mRNA is produced from each genes or group of genes that is to be

expressed.iv) mRNA is a very heterogeneous class of molecule. They correctly assumed

that three nucleotides code for one amino acid.v) The messenger should be synthesized and degraded very rapidly.vi) In E.coli, the average length of an mRNA is about 1.2kb.

2. Transfer RNA(tRNA):i) Carries amino acids in the activated form to the ribosome for peptide bond

formation, in such a sequence that can be determined by the mRNA template.

ii) There is at least one kind of tRNA for each of the 20 amino acids.iii) Transfer RNA consists about 75 (approximately 73-93) nucleotides, which

makes it smallest of the RNA molecule.3. Ribosomal RNA(rRNA):

i) It is the major component of ribosome.ii) It plays both a catalytic and structural role in protein synthesis.iii) rRNA is the most abundant of the three types of RNA. tRNA comes next,

and then mRNA constitutes only 5% of the total RNA.4. Small nuclear RNA(snRNA):

i) Eukaryotic cells contain additional small RNA molecule that participate in the splicing of RNA exons.

RNA polymerase I, II, III

RNA polymerase I (pol I): that only transcribes rRNA.

RNA polymerase II (pol II): catalyzes the transcription of DNA to synthesize precursor of mRNA and most snRNA and microRNA (22 nucleotides). A wide range og transcription factors are required for it to bind to its promoters & begin transcription.

RNA polymerase III (pol III): transcribes DNA to synthesize tRNA and other small RNA.

Transcription begins near promoter sites and ends at a terminator sites.-Explain.

Promotor: A promoter is a region of DNA that facilitates the transcription of a particular gene. RNA polymerase binds in the promoter site of DNA.

DNA templates contain regions called promoter sites that specifically bind RNA polymerase and determine where transcription begins.

Eukaryotic genes encoding proteins have promoter sites with a TATAAAA sequence centered at about -25 (25 nucleotides on the 5’ side of the first one transcribed). This is called TATA box. Many eukaryotic promoters also have CAAT box with a GGNCAATCT consensus sequence centered at about -75.

Transcription of eukaryotic gene is further stimulated by enhancer sequences which can be quite distant (up to several kilo bases) from the start site.

Termination: RNA polymerase proceeds along the DNA template and transcribes one of its strands until a terminator sequence is reached. That sequence codes for a termination signal, a stable hairpin like structure is formed. This hairpin is formed by base pairing of self complementary sequence that is rich in G & C. The termination is also done by rho protein.

The features of tRNA:

All known tRNA has the following features:

1. Each is a single chain containing between 73 and 93 ribonucleotides.2. They contain many unusual bases, typically between 7 and 15 per molecule. Some are methylated or dimethylated derivatives of A, U, C, and G formed by enzymatic modification of a precursor tRNA. Methylation prevents the formation of certain base pairs, thereby rendering some of the bases accessible for other interactions. In addition, methylation imparts a hydrophobic character to some regions of tRNAs,

which may be important for their interaction with synthetases and ribosomal proteins. 3. About half the nucleotides in tRNAs are base-paired to form double helices. Five groups of bases are not base paired in this way: The 3’ CCA terminal region, which is part of a region called the acceptor stem; The TψC loop, which acquired its name from the sequence ribothymine-

pseudouracil-cytosine; The "extra arm," which contains a variable number of residues; The DHU loop, which contains several dihydrouracil residues; The anticodon loop. The structural diversity generated by this combination of helices and loops containing modified bases ensures that the tRNAs can be uniquely distinguished, though structurally similar overall.

4. The 5’ end of a tRNA is phosphorylated. The 5’ terminal residue is usually pG.

5. The activated amino acid is attached to a hydroxyl group of the adenosine residue located at the end of the 3’ CCA component of the acceptor stem. 6. The anticodon is present in a loop near the center of the sequence. This loop contains seven bases.

Restriction Enzyme

Restriction enzyme splits DNA into specific fragments. It is also called Restriction endonuclease. They recognize specific base sequence in double helical DNA and cleave both strands of the duplex at specific places.

They are used for:

1. Analyzing chromosome structure.2. Sequencing very long DNA molecule.3. Isolating genes &4. Creating new DNA molecule that can be cloned.

For example: