RNA: Transcription and Processing Chapter 8

Transcription in Eukaryotes Similar to transcription in prokaryotes but more complicated 3 primary reasons for more complexity Larger eukaryotic genome (more genes) Presence of the nucleus Genomic DNA is organized into chromatin in eukaryotes

Larger Eukaryote Genomes and more RNA pols Gene density in E.coli 1 in 1400bp Gene density in humans 1 in 100,000bp RNA pol I transcribes rRNA gene (exclude 5S rRNA) RNA pol II transcribes all protein-encoding genes (mRNAs and some snRNAs) RNA pol III transcribes the small function RNA genes (tRNA, some snRNA and 5S rRNA) GTFs needed for RNA pol II to bind General Transcription Factors

Nucleus in eukaryotes… RNA processing Modify RNA after it has been transcribed Must remove introns Put exons together Primary transcript (pre-mRNA) differs from processed transcript (mRNA)

Transcription Initiation in Eukaryotes The core of RNA pol II can not recognize its own promoter (similar to bacteria) However, eukaryotes require GTFs to bind to regions in the promoter and not σ GTFs either bind to DNA or other GTFs to recruit/attract RNA pol II GTFs and RNA pol II constitute the preinitiation complex (PIC)

Form the PIC The TATA box is similar to the -35 and -10 region in prokaryotes TBP is part of the TFIID complex (one of six GTF) When TBP is bound to TATA box, it recruits other transcription factors and RNA pol II thus forming the PIC After transcription starts, RNA pol II dissociates from most of the TFs to elongate RNA. In this way, multiple RNA pol II can simultaneously synthezise transcripts from the a single gene

Elongation and termination Elongation takes place inside the transcription bubble (just like bacteria) Destabilization of the RNA–DNA hybrid in the Pol II active site is likely to be a key feature in termination. However RNA in eukaryotes must go through post-transcriptional processing Addition of a 5’cap (Guanylyl transferase adds guanine cap) Splice to remove introns Addition of a 3’ poly A tail (polyadenylation) Experiments have NOW shown that processing takes place cotranscriptionally What does this mean?

Cotranscriptional Processing

Cotranscriptional processing of RNA Poly A tail and 5’methylated cap

The functional equivalent of the TATA binding protein in prokaryotes is: A) a sigma subunit. B) a holoenzyme. C) the Rho factor. D) TFIID.

1 minute paper What have we cover today? What do I need to review about transcription? Differences between prokaryotic and eukaryotic transcription…

RNA splicing The removal of introns Occurs in the mRNAs Discovered in 1977 Occurs in the mRNAs There is also something call alternative splicing

Intron Removal and Exon Splicing GU –AG rule: Each intron is cut at each end and these intron ends almost always have GU at the 5’ end and AG at the 3’ end A residue (the branch point A) between 15 and 45 nucleotides upstream of the 3’ splice site

Spliceosome assembly and function

Reactions in exon splicing

Alternative Splicing 101 TMBr-1 - Tropomyosin is a two-stranded alpha-helical coiled coil protein found in cell cytoskeletons. (Brain specific) TMBr-2 Fibroblasts - A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Smooth muscle - muscle tissue in which the contractile fibrils are not highly ordered, occurring in the gut and other internal organs and not under voluntary control Striated muscle - muscle tissue in which the contractile fibrils in the cells are aligned in parallel bundles, so that their different regions form stripes visible in a microscope. Muscles of this type are attached to the skeleton by tendons and are under voluntary control.

Small Functional RNAs that Regulate and Protect the Eukaryotic Genome Lin-4 in C. elegans Codes for a 22nt RNA product – first miRNA identified DICER Enzyme Recognizes dsRNA and cleaves them into 22nt products dsRNA Considered foreign in eukaryotes Triggers a defense response/immune reaction RISC RNA induced silencing complex

miRNA halt translation of target genes Lin-4 represses Lin-14 and Lin-28 Affects development of C.elegans

Three experiments demonstrating gene silencing

Three experiments demonstrating gene silencing Petunia flowers demonstrating co-suppression

Three experiments demonstrating gene silencing

Ways to generate dsRNA

siRNAs degrade mRNA from viral genes or transposons

Concept Check Template strand DNA and encoded RNA are: A) complimentary of one another with antiparallel orientation. B) complimentary of one another but share the same 5′–3′ orientation. C) identical sequences with the exception of U substituted for T in DNA. D) different with regard to the inclusion of exons.

Can you explain this picture?