Biosynthesis of RNA - Transcription:

With exception of biosynthesis of RNAs in such organelles as mitochondria and chloroplasts, in eukaryotic cells site of DNA dependent. RNA biosynthesis (transcription) is nucleus, while nucleus seems to have enzymes and genes for ribosomal RNA (rRNA) biosynthesis, the enzymes liable for synthesis of transfer RNA (rRNA) and messenger RNA (mRNA) are localized in nucleoplasm. In prokaryotic organisms, RNA polymerase happens in cytoplasm. Biosynthesis of ribonucleic acid needs presence of enzyme RNA polymerase, magnesium ions and triphosphate ribonucleotides. Another very necessary need is presence of nucleic acid (DNA or RNA) template. Unless there is nucleic acid template present, polymerase will create RNA with random arrangement of nuclieotide bases. It is chromosomal DNA that normally gives template for RNA formation in most cells.

DNA as Template for RNA Transcription:

Though the nucleotide sequence in DNA helix was said to be random, it should be emphasized that for each species of organism, nucleotide sequence on DNA is extremely particular for all cells inside organism; by random is only meant that there is no rule as to which bases takes place in whatever sequence. DNA acts as template for making, i.e., transcription of RNA. DNA first unzips, with two strands separating because of breakage of hydrogen bonds between them. Though, it is only one of the strands which serve as template for transcription and is called as sense strand while other is called as Non-sense or anti-sense strand. Therefore in transcription, a complementary strand of RNA is created to sense strand of DNA double helix. Process of transcription is mediated by RNA-polymerase, a DNA-dependent enzyme that attaches to DNA molecule and catalyzes formation of phosphate between ribonucleotides.

Association with DNA Template:

RNA biosynthesis doesn't need template prime as in DNA replication. Transcriptioon starts at specific promoter sites in DNA template and terminates at the end of defined sequence presumably RNA core polymerase frequently relates and dissociates with DNA template until promoter site is found; promoter sites should have specific sequence which are recognized by holo - RNA polymerase as appropriate binding site. Sigma factor is necessary component for binding at promoter site. With G factor, only sense strand will be identified and read properly. As the 5' end of many RNAs has either PPP A or PPPG, either ATP or GTP is possibly bound originally by RNA polymerase at promoter site. Sigma (σ) factor is required for initiation of transcription but once began it dissociates and core RNA polymerase completes transcription. Rate of transcription is apparently controlled by rate of initiation and not be rate of elongation.

Termination:

At the end of gene, a sequence of bases should signal completion of transcription. Release factor, rho (p), oligomeric protein, with molecular weight of 200,00, apparently joins to RNA polymerases blocking further transcription and RNA product is released.

Transcription in Eucaryotes:

Eucaryotic cells have at least three various nuclear RNA polymerases. RNA polymerase I, an Oligomeric protein found in nucleus, synthesizes ribosomal RNA (rRNA); it has molecular weight of 500,000 - 700,000 and needs either Mg2+or Mn2+. RNA polymerase II is oligomeric protein found in nucleoplasm; it needs Mn2+ for synthesis of messenger RNA (mRNA). RNA polymerase III, also found in nucleoplasm is liable for synthesis of transfer RNA (tRNA). Polymerase IV is localized in inner mitochondrio membrane and is concerned with asymmetric transcription of mitochondrial ribosomes. In higher plants, that have chloroplasts, a DNA-dependent RNA polymerase has been classified. All these polymerases in eukaryotic cells have G like initiation factors related with them.

Post - Transcriptional Processing of RNA:

Newly synthesized single-stranded RNA may suffer a number of alterations. Transcription results in synthesis of precursors for mRNA, rRNA and tRNA that are altered if essential, and transported to cytoplasm where they function. Nuclear RNA's can be divided somewhat randomly in four classes, on the basis of size, rate of turn-over, sequence difficulty and function. Relative abundance of these classes varies with cell type and development conditions. Class termed heterogenous nuclear RNA (hnRNA) is usually the most abundant. These molecules range from approx 1,000 to 50,000 nucleotides in length. Processing of mRNA by cleavage also has been suggested on basis of differences between bulk hnRNA and cytoplasmic RNA. These two RNA populations illustrate different, although overlapping size distributions in higher eukaryotes. mRNA chain lengths range from approx 1,000 to 10,000 nucleotides, while hnRNA chain lengths appear to range from 1,000 to 50,000 nucleotides. Therefore, in eukaryotic cells a prescursor, heterogenous nuclear RNA (hnRNA) is first synthesized in nucleoplasm by DNA-dependent RNA polymerase. It is then degraded by the nuclear nuclease to mRNA that is then translocated to cytoplasm. Where it becomes related with string of ribosomes. Most eukaryotic mRNAs are monocistronic, that is, code for only one polypeptide. Precursor tRNA also passed out from nucleus in cytoplasm through nucleoplasses. In cytoplasm it then suffers secondary folding in its typical clover-shape form. This structure is helped by hydrogen bondings among bases which do overlap; anticodon is situated in central petal of clover leaf. Ribosomes are composed of two subunits that enter cytoplasm through pores in nuclear envelope and are in fundamentally nascent (complete) forms. On analysis, ribosomes of bacteria contain approx 60% RNA and 40% protein by weight. No lipids or polysaccharides are found in bacteria ribosomes. Eucaryotic ribosomes have smaller proportion of RNA with ration of RNA; protein approaching value of 1that is RNA protein = 1:1. Protein complements of ribosomes are very complex. Bacteria ribosomes may have 50 to 60 different proteins; eukaryotic ribosomes show greater complexity and may have as many as 150 different polypeptide chains. Three classes of ribosomal proteins exist as stated by their strength of bonding to ribosomes. In prokaryotes holding together of ribosomal units depends on concentration or Mg²⁺_. Approx 30% of protein is only very loosely joined and is easily removed; removal of protein doesn't interfere with protein synthesis in cell - free systems.

Another factor which is approx 50% of proteins can be reversibly removed by adjusting concentration of Mg²⁺. The remaining 20% known as core protein is very tightly bound, its removal results in irreversible destruction of ribosome.

At levels of 0.005m to 0.01m Mg²⁺, approx 30% of the protein is only very loosely joined and is easily removed; the removal of this protein doesn't interfere with protein synthesis in cell-free systems. Another fraction that is about 50% of proteins can be reversibly removed by adjusting concentration of Mg²⁺. The remaining 20%, known as core protein is very tightly bound, its removal results in irreversible destruction of ribosome. At levels of 0.005m to 0.01m Mg²⁺, bacterial ribosome remain intact. At levels below 1,000 Mg²⁺ ions/ribosome, ribosomal sub units are irreversibly denatured if "core" proteins are removed. Eucaryotic ribosomes aren't denatured by just low Mg²⁺ concentration but you have to raise PH or increase concentration of monovalent cations or increase concentration of PO₄ or CO₃ which have high affinity for Mg²⁺. These observations point to possible role of Mg²⁺ ions in binding the subunits of ribosomes together.

ibosomes in both prokaryotic and eukaryotic cells are made up of two sub-units, one smaller than the other. It has also been illustrated that protein synthesis in mitochondria and chloroplasts is inhibited by chloroamphinicol, an inhibitor that is effective against bacterial protein synthesis in eukaryotic cells. Chloroamphenicol has no effect on protein synthesis in cytoplasm (i.e. on ribosomes) in eukaryotic cells.

Differences between RNA, DNA and their Syntheses:

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