Karp_Ch11_LecSlides_2of3.ppt

11.4 Synthesis and Processing of Messenger RNAs (1) • The precursors of mRNAs are represented by diverse RNAs called heterogeneous nuclear RNAs (hnRNAs). – hnRNAs are found only in the nucleus. – hnRNAs have large molecular weights. – hnRNAs are degraded after a very short time. The formation of...

11.4 Synthesis and Processing of Messenger RNAs (1) • The precursors of mRNAs are represented by diverse RNAs called heterogeneous nuclear RNAs (hnRNAs). – hnRNAs are found only in the nucleus. – hnRNAs have large molecular weights. – hnRNAs are degraded after a very short time. The formation of hnRNA and its conversion into smaller mRNAs Synthesis and Processing of Messenger RNAs (6) • The Structure of mRNAs: Messenger RNAs share certain properties – They each code for a specific polypeptide. – They are found in the cytoplasm. – They are attached to ribosomes when translated. – Most have a noncoding segment. – Eukaryotic mRNAs modifications at their 5’ (guanosine cap) and a 3’ poly(A) tail. Structure of the human -globin mRNA Synthesis and Processing of Messenger RNAs (7) • Split Genes: An Unexpected Finding – The difference between hnRNA and mRNA provided early clues about RNA processing. – Eukaryotic genes contain intervening sequences which are missing from mature mRNAs. – The presence of genes with intervening sequences are called split genes. The difference in size between hnRNAs and mRNAs Synthesis and Processing of Messenger RNAs (2) • The Machinery for mRNA Transcription – RNA polymerase II is assisted by general transcription factors (GTFs) to form the preinitiation complex (PIC). – The critical portion of the promoter lies 24-32 bases upstream from the initiation site, and contains the TATA box. – The preinitiation complex of GTFs and polymerase assemble at the TATA box. Initiation of transcription from a eukaryotic polymerase II promoter Initiation of transcription from a eukaryotic polymerase II promoter Synthesis and Processing of Messenger RNAs (3) • The preinitiation complex assembly starts with the binding of the TATA-binding protein (TBP) to the promoter. • TBP is a subunit of the TFIID and when it binds to the promoter causes a conformation change in DNA. Structural models of the formation of the preinitiation complex Synthesis and Processing of Messenger RNAs (4) • Binding of TFIID sets the stage for the assembly of the complete PIC. • The three GTFs bound to the promoter allows the binding of RNA polymerase with its TFIIF. • As long as TFIID remains bound to the promoter, additional RNA polymerases may be able to attach for additional rounds of transcription. Initiation of transcription by RNA polymerase II Synthesis and Processing of Messenger RNAs (5) • RNA polymerase is heavily phosphorylated at the carboxylterminal domain (CTD). • CTD phosphorylation can be catalyzed by different protein kinases. • TFIIH acts as the protein kinase. • Termination of transcription is not well understood. The discovery of intervening sequences Synthesis and Processing of Messenger RNAs (8) • The parts of the split gene that contribute to the mature mRNA are called exons. • The intervening sequences are called introns. • The discovery of genes with introns led to investigate how these genes were able to produce mRNAS lacking these sequences. Synthesis and Processing of Messenger RNAs (9) • Hybridization experiments supported the concept of mRNA precursors (pre-mRNAs). • Loops in the DNA-RNA complex were the introns. • The loops resulted from introns that were not complementary to any part of the gene. Visualizing an intron in the globin gene Visualizing introns in the ovalbumin gene Synthesis and Processing of Messenger RNAs (10) • The Processing of Eukaryotic Messenger RNAs – RNA transcripts become associated with ribonucleoproteins as they are synthesized. – During processing, a 5’ methylguanosine cap and 3’ poly(A) tails are added. – Intervening sequences are removed and exons are connected by RNA splicing. Pre-mRNA transcripts are processed cotranscriptionally Steps in the addition of 5’ methylguanosine cap and a 3’ poly(A) tail to a pre-mRNA Synthesis and Processing of Messenger RNAs (11) • RNA Splicing: Removal of Introns from a Pre-mRNA – Breaks are introduced at the 5’ and 3’ ends (splice sites). – Sequences between exon-intron boundaries are highly conserved. – Sequence most commonly found at the boundary is g/GU at the 5’ end and AG/G at the 3’ end. Nucleotide sequences at the splice sites of pre-mRNAs Synthesis and Processing of Messenger RNAs (12) • The mechanism of RNA splicing has led to the study of RNA enzymes, or ribozymes. • RNA splicing is thought to have evolved from self-splicing RNAs. • An example of a self-splicing intron is the group II intron, discovered in various organisms. The structure and self-splicing of group II introns Synthesis and Processing of Messenger RNAs (13) • The pre-mRNA is not capable of selfsplicing, and requires small nuclear RNAs (snRNAs). • As each hnRNA is transcribed, it becomes associated with a hnRNP. • Processing occurs as each intron becomes associated with a complex called spliceosome. • The spliceosome consists of small nuclear ribonucleoproteins (snRNPs). Model of the assembly of the splicing machinery Synthesis and Processing of Messenger RNAs (14) • Removal of an intron requires: – Several snRNP particles. – Each snRNP contains a dozen or more proteins, such as the Sm protein family. Synthesis and Processing of Messenger RNAs (15) • snRNAs may be the catalytically active components of the snRNPs, not the proteins based on: – Pre-mRNA are catalyzed by the same pair of chemical reactions. – The snRNAs required for splicing pre-mRNA resemble group II introns. • There is a proposal that the combined action of both RNA and a protein in the spliceosome catalyze the two chemical reactions required for RNA splicing. Proposed structural similarity between reactions carried out by spliceosome and self-splicing A mechanism for the coordination of transcription, capping, polyadenylation, and splicing. Processing the ovomucoid premRNA