- each snurp consists of RNA + protein

- U1, U2, U5, and U4/U6 named because rich in uridine residues

- about 40 proteins (Sm) in U1 - U6

- the snRNA components of the splicing apparatus interact both among themselves and with the substrate RNA by means of base pairing interactions.

- the spliceosome performs splicing reactions by means of a catalytic centre
 

Other splicing reactions

- 4 classes of introns in protein coding genes

(1) nuclear pre-mRNA in higher eukaryotic genes

(2) Group I organelles, bacteria and

(3) Group IIA lower eukaryotes

(4) GroupIV e.g. in tRNA of yeast

- in Group I and II introns the RNA is AUTOSPLICING - it performs the splicing reactions by itself without need for enzymatic activities of proteins
 

- in vitro, rxns can be performed by RNA alone but is assisted by proteins in vivo

these properties are intrinsic and due to specific conformation of molecule

- RNA molecules that function as enzymes called
 

Group I introns

e.g. Tetrahymena thermophila rRNA precursors

- also common in transcripts of fungal mitochondria genes

- requires guanosine nucleoside or nucleotide with a free OH group

Fig

Group II introns

- reaction in mitochondria and chloroplasts similar to that of nuclear splicing - via a lariat held together by a 5'-2' phosphodiester bond

- splice sites resemble those of nuclear pre-mRNA

- phosphodiester bonds conserved, so no external energy source required
 
 

Group IV introns

e.g. tRNA precursors in Saccharomyces cerevisiae

splicing depends on recognition of a common secondary structure (folding) rather than on consensus sequences in the intron

2Estructure due to base pairing between sequences in the intron and exon

splicing endonuclease cleaves the precursor at both ends of the intron giving rise to a linear intron and two half-tRNA molecules

(folding)

RNA ligase joins the halves covalently with a phosphodiester bond - this reaction requires ATP in vitro

The reactions involved:

one end of the half-tRNA ends in a 5'OH, the other is marked by a 2'-3' cyclic phosphate group

the cyclic phosphate group is opened to generate a 2' phosphate and a 3' OH
The 5'OH generated by the splicing nuclease is phosphorylated to give a 5' phosphate

the halves refold to bring the two ends of the splicing junction into close proximity

splicing ligase can now join the 5' phosphate at one end to the 3'OH at the other end of the splicing junction
 
 

Summary

in nuclear RNA and in groups I and II introns splicing proceeds by two transesterifications

-(1) a free OH group attacks the phosphodiester bond at the exon-intron junction

- (2) the OH created at the end of exon 1 attacks the phosphodiester bond at the intron-exon 2 junction
 
 

Enhancers

a cis-acting sequence that increases utilisation of (some) eukaryotic promoters

can function in either orientation and in any location (upstream or downstream) relative to the promoter

(similar to UAS in prokaryotes except UAS always upstream)

contains several closely arranged sequence elements that bind transcription factors

may be responsible for tissue-specific transcription e.g. Ig genes that carry internal enhancers which are active only in the B lymphocytes on which the Ig genes are expressed

enhancers appear to function by localization of the protein bound at the enhancer to increase its chance of contacting proteins that bind the promoter

this may necessitate DNA folding or looping as enhancers are often hundreds of bp from the promoter
 

• Syllabus

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