RNA MODIFICATION Eukaryotic mRNA molecules are modified before they exit the nucleus.

Alteration of mRNA 5’ end As soon as the mRNA is transcribed modifications occur The 5’ end is immediately capped of with a modified Guanine - a 5’ CAP It has 2 functions – Protects mRNA from hydrolytic enzymes – Part of recognition site for ribosome attachment

Alteration of mRNA 3’ end The 3’ end is also modified An enzyme adds a “poly A” tail nucleotides It has some similair functions – Protects mRNA from hydrolytic enzymes – Part of recognition site for ribosome attachment Also facilitates export of mRNA from nucleus

Nonsense or Selfish DNA Most genes of complex eukaryotes are interrupted by long noncoding segments which are called intervening sequences (introns).

Exons and Introns Average length of a transcription unit is 8000 nucleotides. But coding region is approx 1200 nucs Coding regions (exons) are interrupted by introns Both exons and introns are transcribed into RNA. Before the RNA leaves the nucleus, the introns are removed and the exons are rejoined to produce a coding sequence of mRNA.

RNA Splicing This process, called RNA splicing or RNA processing, is also required for tRNA and rRNA production. Signals for RNA splicing are sets of a few nucleotides located at either end of each intron Particles called snRNPS recognize these splice sites snRNPS are part of spliceosomes

snRNP’s Small nuclear ribonucleoproteins (snRNPs). These are small particles located in the cell nucleus that are composed of RNA and protein molecules. The RNA in snRNPs is called small nuclear RNA (snRNA).

“snurps” snRNA is a single molecule about 150 nucleotides long (twice as long as tRNA but shorter than mRNA). Each snRNP contains seven or more proteins. Several types of snRNPs have been discovered but their functions have only been partly determined.

Spliceosome Those kinds of snRNPs known to participate in RNA splicing function as components of a larger, more complex unit called a spliceosome. Spliceosomes interact with the ends of RNA introns by cutting at specific points to release the introns. The two exons which flanked the removed intron are rejoined into adjacent codons.

Role of Introns Some Introns contain gene regulation sequences which may control the passage of mRNA into the cytoplasm There also seems to be a correlation between complexity and the number of Introns. The more intros that separate the coding exons the more opportunity for “mixing and matching” of exons. This process is known as alternative splicing.

Domains Many proteins consist of various “domains” -for example the active site may be its own domains Each exon of the split gene codes for a domain Some domains are highly conserved, some act as “cassette” exons and can be switched in and out Thus one gene can be responsible for the production of 100’s if not 1000’s of proteins through this alternative splicing technique.

Post translational modification Once the translated polypeptide chain has been produced it folds up spontaneously However several modifications may be required to form a functional protein Amino acids may be modified by addition of sugars, lipids, or phosphate groups The protein may be cleaved in two These processes and the the variety of post translational modification that can occur further increase the variety of functional proteins a gene can generate

Wobble Theory Crick suggested that the base in the third position of the anticodon does not have as strict base-pairing requirements as the other two base pairs This allows it to form hydrogen bonds with several bases at the 3' end of the codon. Especially true when the base in this third position is inosine (abbreviated I)

Wobble Hypothesis The genetic code is degenerate. This means that slightly different structures can perform the same function. In this case more than one codon can give the same amino acid – the codons are degenerate. Example, CCU, CCA, CCC, and CCG all encode proline. The end result is that 61 codons encod ethe 20 amino acids. Does this mean we need 61 different tRNA’s?

Wobble Hypothesis No – only about 40 or so are needed due to the 3 rd base in the anti codon being “wobbly” Technically this means that the base pairing rules are not strictly observed in this position This phenomena is called “Wobble” Ex – it allows GCA,GCU and GCC to be recognized by the SAME tRNA It is important to recognize that it does not allow for ambiguity

Wobble Hypothesis No Ambiguity: each mRNA codon always results in the same amino acid the tRNA bringing the AA may differ in its anticodon due to wobble but the codon always specifies the same AA

Inosine Inosine is only found in tRNA and only in the third position It is particularly "wobbly” and has more flexibility in it base pairing Because of the wobble hypothesis, fewer than 61 tRNAs are required to recognize all of the possible sense codons.

Wobble Base Pair Rules 3rd Position of tRNA anticodon 3 rd position of matching codon GC or U CG AU UA or G IA or U or C