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Regulation of Gene Expression (Continued)

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Biochemistry 2280A
Derek Mc Lachlin

Brandl Lecture 3 Notes 11/12/2012 Formation of mRNAs: RNA Processing in Eucaryotic Cells  Primary transcript becomes a mRNA for export out of the nucleus and translation  Three main steps: o 5’ capping o 3’ polyadenylation o Splicing  Each requires a specific enzyme(s) 5’ Capping  When RNA is made, first nucleotide added is a triphosphate, and a 7-methylguanosine is added to it to the 5’ end through a unique 5’-5’ linkage  Marks the 5’ end of the mRNA as being intact  Required for mRNA export from the nucleus  Required for translation of the mRNA into protein (translational initiation) 3’ Polyadenylation  Happens while transcription is ongoing  Sequence that signals polyadenylation, endonuclease cleavage and addition of polyA tail  Helps protect the 3’ end of the mRNA from degradation  Indicates that the 3’ end of the mRNA is intact and therefore is important for: o Export out of the nucleus o Translation RNA Splicing  In eucaryotic cells, protein encoding sequences are interrupted by one or more noncoding sequences called introns  Coding sequences are called exons  Exons = expressed, introns = interruptions  Introns are spliced out of the primary transcript to give the mature RNA  The splicing pattern is often tissue specific Why is Splicing Important?  Differential splicing – a single RNA can be spliced in different ways to create related but distinct proteins  Splicing enhances the coding capacity of the genome – each gene can make more than one protein Mechanism of Splicing  Splicing requires specific sequences in the RNA – it is RNA that triggers splicing  Three key sequences: o 5’ splice junction – between exon and intron o 3’ splice junction – between exon and intron o Branch point – adenine must be present in branch point – essential for splicing  Introns are removed in two consecutive transesterification reactions 1. Attack by the 2’-OH of the adenine at the branch point with the 5’ splice junction – releases 5’ exon and creates lariat structure 2. Attack by the 3’-OH of the free 5’ splice junction with the 3’ splice junction – releases the lariat and joins the two exons Spliceosome  The enzymatic machinery for splicing  Composed of snRNPS, U1, U2, U4, U5, and U6  snRNPS contain both RNA and protein – RNA plays a key role in the splicesome as it holds thing together and is fundamental for molecular recognition  Through RNA-RNA and protein-protein interactions, the splices
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