1.Structure and chemistry of nucleic acids – RNA (rRNA, snRNA).
rRNA molecules are the structure components of ribosome and they are produces after transcription by RNA polymerase III of 300-400 tandem repeats organized in five cluster. They assist in protein synthesis by combining with protein in the cytoplasm to form ribosome and acts as the site of protein synthesis. The rRNA contain a number of specially modified nucleotides, including pseudouridine, ribothymidylic acid, and methylated bases. rRNA form into two subunits; small subunits of rRNA reads the order of amino acids and large subunits links the amino acids and forming peptide bond.
snRNA has a range in size about 80 to 350 nucleotides and approximately 150-nt-long ncRNAs. They are components of spliceosomes which contains 145 proteins and found in stable complexes with specific proteins forming small nuclear ribonucleoprotein particles, or snRNPs, which are about 10S in size. In eukaryotes, they are found in nucleus and their relative abundance (1% to 10% of the number of ribosomes) are significant clues to their biological purpose. They involve in pre-mRNA processing, regulation of RNA polymerase II and maintenance of telomeres.
2. Small RNA (siRNA, miRNA, snoRNA)
siRNA has two RNA strands, antisense strand and sense strand which then form a duplex 21 bp long with 3′ dinucleotides overhang on each strand. They consist of 19 to 23 nucleotides long by base pairing with complementary sequences within a particular mRNA to form regions of double-stranded RNA (dsRNA) and dsRNA region are specifically degraded, eliminating the mRNA from the cell. siRNA operates within RNA interference (RNAi) pathway, where its disrupts gene expression by blocking specific protein production, even though the mRNA encoding the protein has been synthesized. This is known as gene silencing due the expression of the specific gene is silence.
miRNAise short, single-stranded RNA, non-coding which consists of 22 nucleotides in length and with two nucleotides at 3′ end. Its functions involve in many biological process such as cell cycle control, apoptosis, several developmental and physiological processes and helps in differentiation and maintenance of tissue identity. In gene silencing, miRNA bind to 3′ UTR (untranslated region) of the target mRNA. This prevent the protein production by destabilizing the mRNA and translation silencing.
snoRNA functions are involved in modification and processing or ribosomal RNA (rRNA) and some are used to modify snRNA, mRNA and tRNA. They consists of 60 to 300 nucleotides long and located in nucleolus.
4. Endo/Exo-Nuclease with Type I, II, III
Type I is an enzyme that has 3 subunits for restricted digestion and methylase activity. It cuts nucleotides at the site away from the recognition site with the help of ATP and S-adenosyl-methionine (SAM).
Type II endonuclease means there are two different enzymes, one is to cleave the nucleotides, one is to modify the recognition sequence. They do not use ATP for their activity. It cleaves the sequence at a short specific distance within or from the recognition site.
Type III is a single enzymes that contains two subunits. They cut at a site that is close to the identification site. Same with the type I, they also require ATP.
With the help of Type I exonuclease, the single-stranded DNA is broke in a 3′ to 5′ direction. The phosphoryl group of the DNA strand needs to be first unblocked. If not, the chains cannot be cleaved because they do not have terminal 3′-OH groups.
Type II exonuclease is linked with DNA polymerase I. DNA polymerase I contain a 5′ exonuclease that helps in cutting RNA primers directly upstream of the DNA synthesis site in a 5′ to 3′ direction.
Type III exonuclease catalyzes the removal of single nucleotides, one by one, from the 3′-OH end of the polynucleotide chain.