Molecular biology

splicing event

splicing process:
introns interrupt the protein coding region of mRNA. intron sequences occur in DNA and transcribed into pre-mRNA. introns must be removed accurately. splicing rection is catalysed by SNURPS and takes plase in a complex called spliceosome. spliceosome complex consists of pre-mRNA and several different SNURPS. mature mRNA only exits nucleus after splicing process is complete. splice sites are specified by sequence at the ends of the introns at the end of introns at the 5' and 3' splice junctions. GU-AG rule is constant in all splicing process involving spliceosome.

transesterification:
step 1. 2'OH group of adenosine nucleotide in the intron attacks the phosphodiester bond at 5' splice site. bonds breaks and new phosphodiester bond is formed.
step2. 3'OH group of free 5' exon attacks 3' splice site. net result of this is the exons are joined and introns are released as branched lariat structure.
since the number of phosphodiester bond is constant, no energy is used in this process.

self splicing introns pure: pure preparations of some RNA transcripts slowly splice the introns IN THE ABSENCE OF ANY PROTEINS.

group I introns: this occurs in nuclear rRNA genes in protozoan. the event involves the 2 transesterifications. role of 3'OH of G cofactor is similar to that of 2'OH of the branch point adenosine in spliceosomal mechanism. but G cofactor is not part of RNA chain i.e it is not carried together like the spliceosomal machinery components.

group II introns: this group of introns occur in mitochondria and chloropast in plants and fungi. introns fold into conserved secondary structure containing stem loops. the splicing mechanism also involves the 2 transesterifications similar in spliceosomal splicing. SNURPS in spliceosome functions similar to the stem loops in group II introns. thus it is hypothesised that spliceosome mechanism probably had evolved from self-splicing mechanism. secondary structures of group II self splicing introns and U SNURPS present in spliceosome. maturases bind to group II intron and increase the rate of splicing. maturases is said to stabilise the 3D structure of intron while SNURPS is said to stabilise the structures in spliceosomal splicing.




DNA topology

the great majority of DNA in living cells occurs as B form. there are certain flexibilities within B form. 1. the number of base pairs per turn of helix can be altered. 2. helix in the cell is not straight, but rather coil in 3D space. 3. there are certain sequence feature where bends occur. 4. transcription and replication require strand separation.

supercoiling: the DNA in the cell is coiled in 3D and this introduces torsional stress into the molecule. this is known as supercoiling, which can be either negative or positive: negative supercoiling is when the twist of the DNA is opposite to the right hand of the helix. positive supercoiling is when the twist of supercoiling is in the same direction as the turn of the helix.

the torsional stress can be accomodated in 2 ways: 1. formation of superhelices 2. altering the number of base pairs per turn of helix. these 2 possible responses can be incorporated into a single concept; the linking number, L or the total number of times that the strands cross each other in a plane. formula that can be use L=W+T where W is the writhing number, corresponds to the superhelicity and T is the the twisting number, the helical winding measurement.

topoisomerases: this is an enzyme that can alter the number of DNA molecules and there are 2 types of topoisomerase:

Type 1 topoisomerase: this type breaks one strand of the DNA, pass the other strand through and gap and seal the break. linking number, L changes by +/- 1.

Type 2 topoisomerase: this type breaks both strands of the DNA and pass ANOTHER PART OF HELIX through the gap and seal the break. the linking number change by +/-2. example of this type of topoisomerase is DNA gyrase in DNA replication.