Transcription 11/3/05

Stable RNA rRNA -Structural component of ribosomes tRNA-Adaptors, carry aa to ribosome Synthesis Promoter and terminator Post-transcriptional modification (RNA processing) Evidence Both have 5’ monophospates Both much smaller than primary transcript tRNA has unusual bases. EX pseudouridine

tRNA and rRNA Processing Both are excised from large primary transcripts 1º transcript may contain several tRNA molecules, tRNA and rRNA rRNAs simply excised from larger transcript tRNAs modified extensively 5. Base modifications Both are excised from large primary transcripts 1º transcript may contain several tRNA molecules, tRNA and rRNA rRNAs simply excised from larger transcript EX. Transcription of the rrnD operon yields a 30S precursor, which must be cut up to release the three rRNA and three tRNAs. tRNAs modified extensively EX. tRNATyr Endonuclease cleaves ~200 bases from 3’ end RNase D removes 7 bases from 3’ end RNAse P generates 5’ end, leaving a monophosphate RNase D generates 3’ CCA end Extensive modification of bases, all in or near the loops

Examples of Covalent Modification of Nucleotides in tRNA N6-Methyladenylate (m6A) N6-Isopentenyladenylate (i6A) Inosinate (I) 7-Methylguanylate (m7G) tRNA modified bases Dihydrouridylate (D) Pseudouridylate (Ψ) (ribose at C-5) Uridylate 5-oxyacetic acid (cmo5U) 3-Methylcytidylate (m3C) Modifications are shown in blue. 5-Methylcytidylate (m5C) 2’-O-Methylated nucleotide (Nm)

Eukaryotic Transcription Regulation very complex Three different pols Distinguished by -amanitin sensitivity Pol I—rRNA, least sensitive Pol II– mRNA, most sensitive Pol III– tRNA and 5R RNA moderately sensitive Each polymerase recognizes a distinct promoter Regulation very complex Three different pols distinguished by -amanitin sensitivity Pol I—rRNA least sensitive Pol II– mRNA most sensitive Pol III– tRNA moderately sensitive Each polymerase recognizes a distinct promoter Promoters recognized Pol I—bipartite promoter Pol II– Upstream promoter Pol III—internal promoter

Eukaryotic RNA Polymerases Location Products -Amanitin Sensitivity Promoter I Nucleolus Large rRNAs (28S, 18S, 5.8S) Insensitive bipartite promoter II Nucleus Pre-mRNA, some snRNAs Highly sensitive Upstream III tRNA, small rRNA (5S), snRNA Intermediate sensitivity Internal promoter and terminator -amanitin used to distinguish different polymerases. Poison derived from mushrooms (Amanita sp.). How does this explain the reason mushrooms poison the way they do?

Eukaryotic Polymerase I Promoters RNA Polymerase I Transcribes rRNA Sequence not well conserved Two elements Core element- surrounds the transcription start site (-45 to + 20) Upstream control element- between -156 and -107 upstream Spacing affects strength of transcription RNA Polymerase I Transcribes rRNA Sequence not well conserved Architecture well conserved Two elements Core element- surrounds the transcription start site Upstream control element- ~ 100 bp upstream

Eukaryotic Polymerase II Promoters Much more complicated Two parts Core promoter Upstream element TATA box at ~-30 bases Initiator—on the transcription start site Downstream element-further downstream Many natural promoters lack recognizable versions of one or more of these sequences Much more complicated Two parts Core promoter Upstream element TATA box at ~-30 bases Initiator—on the transcription start site Downstream element-further downstream Many natural promoters lack recognizable versions of one or more of these sequences

TATA-less Promoters Some genes transcribed by RNA pol II lack the TATA box Two types: Housekeeping genes ( expressed constitutively). EX Nucleotide synthesis genes Developmentally regulated genes. EX Homeotic genes that control fruit fly development. Specialized (luxury) genes that encode cell-type specific proteins usually have a TATA-box

mRNA Processing in Eukaryotes Primary transcript much larger than finished product Precursor and partially processed RNA called heterogeneous nuclear RNA (hnRNA) Processing occurs in nucleus

Capping mRNA 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA. Guanosine cap is methylated. First and second nucleosides in mRNA may be methylated 1. 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA. 2. Guanosine cap is methylated. 3. First and second nucleosides in mRNA may be methylated What is the function of the 5’ cap? Clue: some viral mRNAs are not capped, yet they are translated efficiently and are stable. In these virus-infected cells the mechanism for initiation of mRNA translation is altered so that capped mRNAs are not translated efficiently! BACK

Polyadenylation Polyadenylation occurs on the 3’ end of virtually all eukaryotic mRNAs. Occurs after capping Catalyzed by polyadenylate polymerase Polyadenylation associated with mRNA half-life Histones not polyadenylated Polyadenylation occurs uniquely on mRNA, not tRNA or ribosomal RNA. This allows simple and rapid purification of mRNA by hybridization to an affinity resin composed of oligo-dT. Histone mRNA is the only non-polyadenylated mRNA in the cell. Why is this??? May be related to histone synthesis uniquely during S-phase of the cell cycle.

Introns and Exons Introns--Untranslated intervening sequences in mRNA Exons– Translated sequences Process-RNA splicing Heterogeneous nuclear RNA (hnRNA)-Transcript before splicing is complete Introns--Untranslated intervening sequences in mRNA Exons– Translated sequences Process-RNA splicing

Splicing Overview Occurs in the nucleus hnRNAs complexed with specific proteins, form a ribonucleoprotein particle (RNP) Primary transcripts assembled into hnRNP Splicing occurs on spliceosomes consist of Small nuclear ribonucleoproteins (SnRNPs) components of spliceosomes Contain small nuclear RNA (snRNA) Many types of snRNA with different functions in the splicing process Occurs in the nucleus hnRNAs complexed with specific proteins, form a ribonucleoprotein particle (RNP) Primary transcripts assembled into hnRNP Splicing occurs on spliceosomes consist of Small nuclear ribonucleoproteins (SnRNPs) components of spliceosomes Contain small nuclear RNA (snRNA) Many types of snRNA with different functions in the splicing process

Splice Site Recognition Introns contain invariant 5’-GU and 3’-AG sequences at their borders (GU-AG Rule) Internal intron sequences are highly variable even between closely related homologous genes. Alternative splicing allows different proteins from a single original transcript Precision is essential in splicing Introns contain invariant 5’-GU and AG-3’sequences at their borders Internal intron sequences are highly variable even between closely related homologous genes.

Simplified Splicing Mechanism 2’ hydroxyl group of an A nt within the intron attacks the phosphodiester bond linking the first exon to the intron. This attack breaks the bond between exon 1 and the intron, yielding the ree exon and a lariat exon-intron intermediate, with the GU linked to the internal A via a phosphodiester linkage to C-2 of ribose. Free 3’ –OH on exon 1 attacks phosphodiester bond between intron and exon 2. Yields spliced exon1-exon2 and lariat shaped intron. Phosphate at end of exon 2 becomes the phosphate linking the 2 exons.

RNA pol III Precursors to tRNAs,5SrRNA, other small RNAs Promoter Type I Lies completely within the transcribed region 5SrRNA promoter split into 3 parts tRNA promoters split into two parts Polymerase II-like promoters EX. snRNA Lack internal promoter Resembles pol II promoter in both sequence and position Precursors to tRNAs,5SrRNA, other small RNAs Promoter Type I Lies completely within the transcribed region 5SrRNA promoter split into 3 parts tRNA promoters split into two parts Polymerase II-like promoters EX. snRNA Lack internal promoter Resembles polII promoter in both sequence and position

DNAse Footprinting Use: promoter ID End Label template strand Add DNA binding protein Digest with DNAse I Remove protein Separate on gel Use: Promoter ID, identification of sequences involved in recognition by proteins End Label template strand Add DNA binding protein Digest with DNAse I Remove protein Separate on gel Run next to sequencing gel (Maxam-Gilbert Sequencing) Will get all sizes represented unless they could not be made because the section was protected by a DNA binding protein. Can focus on region of binding. Protected region