Lecture Molecular biology (Fifth Edition): Chapter 14 - Robert F. Weaver

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In chapter 14, we will see that most eukaryotic genes, in contrast to typical bacterial genes, are interrupted by noncoding DNA. RNA polymerase cannot distinguish the coding region of the gene from the noncoding regions, so it transcribes everything.

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Nội dung Text: Lecture Molecular biology (Fifth Edition): Chapter 14 - Robert F. Weaver

Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 14 RNA Processing I: Splicing Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
mRNA Processing Events • Most eukaryotic genes, in contrast to typical bacterial genes, are interrupted by noncoding DNA • RNA polymerases cannot distinguish the noncoding regions from the coding regions, so it transcribes everything • The cell must remove the noncoding RNA from the primary transcript via splicing • Eukaryotes also add special structures to the 5’ and 3’ ends of the transcript, called the cap and poly-A tail, respectively • All events occur in the nucleus before the mRNA emigrates to the cytoplasm 14-2
14.1 Genes in Pieces • Consider the sequence of the human -globin gene as a sentence: This is bhgty the human globin qwtzptlrbn gene. • Two italicized regions make no sense – Contain sequences unrelated to the globin coding sequences surrounding them – Intervening sequences, IVSs, or introns • Parts of the gene making sense – Coding regions or exons • Some lower eukaryotic genes have no introns 14-3
Evidence for Split Genes • Most higher eukaryotic genes coding for mRNA, tRNA and a few coding for rRNA are interrupted by unrelated regions called introns • Other parts of the gene, surrounding the introns, are called exons • Exons contain the sequences that finally appear in the mature RNA product – Genes for mRNAs have been found with anywhere from 0 to 362 introns – tRNA genes have either 0 or 1 intron 14-4
RNA Splicing • Introns are present in genes but not in mature RNA • How does the information not find its way into mature RNA products of the genes? – Possibility 1: Introns are never transcribed • Polymerase somehow jumps from one exon to another – Possibility 2: Introns are transcribed • Primary transcript result, an overlarge gene product is cut down by removing introns • This is correct process • The process of cutting introns out of immature RNAs and stitching together the exons to form the final product is RNA splicing 14-5
Splicing Outline • Introns are transcribed along with exons in the primary transcript • Introns are removed as the exons are spliced together 14-6
Stages of RNA Splicing • Messenger RNA synthesis in eukaryotes occurs in stages • First stage: – Synthesis of primary transcript product – This is an mRNA precursor containing introns copied from the gene if present – Precursor is part of a pool of heterogeneous nuclear RNAs – hnRNAs • Second stage: – mRNA maturation – Removal of introns in a process called splicing 14-7
Splicing Signals • Splicing must be precise • Splicing signals in nuclear mRNA precursors are remarkably uniform – First 2 bases of introns are GU – Last 2 are AG • 5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifs • Whole consensus sequences are important to proper splicing • Abnormal splicing can occur when the consensus sequences are mutated 14-8
14.2 Mechanism of Splicing of Nuclear mRNA Precursors • Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat • Two-step model – 2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1st exon and G beginning of intron • Forms loop of the lariat • Separates first exon from intron – 3’-OH left at end of 1st exon attacks phosphodiester bond linking intron to 2nd exon • Forms the exon-exon phosphodiester bond • Releases intron in lariat form at same time 14-9
Simplified Mechanism of Splicing • 2’-OH group of A within intron attackes the phosphodiester bond linking the first exon to the intron • A lariat is formed due to the GU at the 5’ end of the intron forming a phosphodiester bond with the branchpoint A • The free 3’OH on exon 1 attacks the phosphodiester bond between the intron and exon 2 • The exons are then linked 14-10
Signal at the Branch • Along with consensus sequences at 5’- and 3’-ends of nuclear introns, branchpoint consensus sequences also occur • Yeast sequence invariant: UACUAAC • Higher eukaryote consensus sequence is more variable • Branched nucleotide is final A in the sequence 14-11
Spliceosomes • Splicing takes place on a particle called a spliceosome • Yeast spliceosomes and mammalian spliceosomes have sedimentation coefficients of 40S and 60S • Spliceosomes contain the pre-mRNA – Along with snRNPs and protein splicing factors – These recognize key splicing signals and orchestrate the splicing process 14-12
snRNPs • Small nuclear RNAs coupled to proteins are abbreviated as snRNPs, small nuclear ribonuclear proteins • The snRNAs (small nuclear RNAs) can be resolved on a gel: – U1, U2, U4, U5, U6 – All 5 snRNAs join the spliceosome to play crucial roles in splicing 14-13
U1 snRNP • U1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequences – U1 snRNA base-pairs with these splice sites – Brings the sites together for splicing is too simple an explanation • Splicing involves a branch within the intron 14-14
Wild-Type and Mutant U1 snRNA • Genetic experiments have shown that base pairing between U1 snRNA and 5’-splice site of mRNA precursor is necessary but not sufficient for binding 14-15
U6 snRNP • U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA • Occurs first prior to formation of lariat intermediate but after first step in splicing • The association between U6 and splicing substrate is essential for the splicing process • U6 also associates with U2 during splicing 14-16
A Model for interaction between a yeast 5’ splice site and U6 snRNA 14-17
U2 snRNP • U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint • This base pairing is essential for splicing • U2 also forms base pairs with U6 – This region is called helix I – Helps orient snRNPs for splicing • 5’-end of U2 interacts with 3’-end of U6 – This interaction forms a region called helix II – This region is important in splicing in mammalian cells, not in yeast cells 14-18
Yeast U2 Base Pairing with Yeast Branchpoint Sequence 14-19
U5 snRNP • U5 snRNA associates with the last nucleotide in one exon and the first nucleotide of the next exon • This should result in the two exons lining up for splicing 14-20