Enzymology of Genetic Engineering || BIOTECHNOLOGY

 Enzymology of Genetic Engineering

In genetic engineering, three types of "Biological tools" are used in the synthesis of recombinant DNA. These include -

i) Enzymes

ii) Vehicle or Vector DNA.

iii) Passenger DNA

          Enzymology or the study of enzymes is an important aspect of genetic engineering. Enzymology is the study of enzymes and its different types used in genetic engineering for the production of different types of matter needed for human well being.

          Many types of specific enzymes are employed in Genetic engineering. These include Lysing enzymes, Cleaving enzymes, synthesizing enzymes, Joining enzymes and alkaline phosphatase:

1. Lysing Enzymes: Lysing enzymes are used to open up the cells to get DNA for genetic experiments. Lysozyme is commonly used to dissolve the bacterial cell wall.

2. Cleaving Enzymes: these are used to break DNA molecules. They are further of three types and these are –

          a) Exonucleases, which cut off nucleotides from 5' or 3' terminal ends of DNA molecule.

          b) Endonucleases: The enzymes cleave DNA duplex at any point except the terminal ends and

          c) Restriction Endonucleases: These cleave DNA duplex at specific point in such a way that single standard free ends project from each fragment of DNA duplex. The single standard DNA free ends are called “Sticky ends” because they can join similar complementary ends of DNA fragment from some other source.

3. Synthesizing Enzymes: These enzymes play a role in the synthesis of DNA strands on suitable templates. They are further of two types –

          a) Reverse Transcriptase, which help in the synthesis of complementary DNA strand on RNA templates.

          b) DNA Polymerase, which help in the synthesis of complementary DNA strand on DNA templates.

4. Joining Enzymes: These enzymes help in sealing gaps in DNA fragments which are otherwise joined by complementary base pairing. Tu ligases are examples. These act as molecular glue. They join DNA fragments by forming phosphodiester bonds.

5. Alkaline phosphatases: These cut off phosphate group from the 5' end of linearised circular DNA to check its recircularization.

 

Messenger RNA or mRNA

The DNA, that controls protein synthesis, is located in the chromosomes within the nucleus, whereas the ribosomes, on which the protein synthesis actually occurs, are placed in the cytoplasm. Therefore, some sort of agency must exist to carry instructions from the DNA to the ribosomes. This agency does exist in the form of mRNA. The mRNA carries the message (information) from DNA about the sequence of particular amino acids to be joined to form a polypeptide, hence its name. It is also called informational RNA or template RNA. The mRNA forms about 5% of the total RNA of a cell. Its molecule is linear and the longest of all the three RNA types. Its length is related to the size of the polypeptide to be synthesized with its information. There is a specific mRNA for each polypeptide. Because of the variation of size in mRNA population in a cell, the mRNA is often called heterogeneous nuclear RNA, or HnRNA.

          A fully processed mRNA has at its 5’ and a cap of methylated guanine followed successively by an Initiation Codon (AUG, GUG), a long coding region, a termination codon (UAA, or UAG or UGA) and a Poly-A tail of many adenine containing nucleotides at 3’ and a small non-coding region, called leader segments, follows the cap and another non-coding region termed Trailer segment precedes the tail. The cap protects the mRNA from degradation by hydrolytic enzymes. It also functions as a part of an “Attach Here” sign for the small ribosomal sub-units. Tail also checks degradation of mRNA and helps the ribosome attached to it. It also helps exist of mRNA from the nucleus. 

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