BMB 400 PART THREE - III = Chpt.12. RNA ProcessingB M B 400, Part ThreeGene Expression and Protein SynthesisChapter 12 RNA PROCESSINGA. Types of RNA processing1. RNA processing refers to any covalent modification to the RNA that occurs aftertranscription. This includes specific cleavage, addition of nucleotides, methylation orother modification of the nucleotides, and removal of introns by splicing.2. OverviewRNAPrecursorModificationAdditionCleavageSplicingmRNApre-mRNA(hnRNA)methylation on2'-OH of ribose5' cap3' poly Acut at site forpoly A; exciseviral mRNAremove intronsrRNApre-rRNAmethylation on2'OH of ribosenoexcise productsfr. precursorremove intronstRNApre-tRNAextensive and variedCCA to 3' endyesremove intronssnRNAs??5' cap??B. Cutting and trimming RNA1. pre-rRNAa. In E. coli, the rrn operon is transcribed into a 30S precursor RNA, containing 3rRNAs and 2 tRNAs.Figure 3.3.1. Excision of rRNAs and tRNAs from 30S precursor RNABMB 400 PART THREE - III = Chpt.12. RNA Processing(1) The segment containing 16S rRNA (small ribosomal subunit) and theone containing 23S rRNA (large ribosomal subunit) are flanked byinverted repeats that form stem structure in the RNA.(2) The stems are cleaved by RNase III. There is no apparent singlesequence at which RNase III cleaves - perhaps it recognizes a particularstem structure. This plus subsequent cleavage events (by an activitycalled M16) generates the mature 16S and 23S rRNAs. The rRNAs arealso methylated.Figure 3.3.2. RNase III cuts in the stems of stem-loops in RNA(3) tRNA is liberated by RNases P and F.(4) 5S rRNA is liberated by RNases E and M5b. In eukaryotes:(1) The initial precursor is 47S and contains ETS1, 18S rRNA, ITS1, 5.8S rRNA,ITS2, and 28S rRNA, where ETS = extragenic transcribed spacer and ITS =intragenic transcribed spacer.(2) Specific cleavage events followed by methylations generate the mature products.Also, some rRNA genes in some species have introns that must be spliced out.2. pre-tRNA in E. colia. Sequence specific cleavage by RNases P, F, D(1) RNase P is an endonuclease that cleaves the precursor to generate the 5' end ofthe mature tRNA.(2) RNase F is an endonuclease that cleaves the precursor 3 nucleotides past the 3'end of the mature tRNA.BMB 400 PART THREE - III = Chpt.12. RNA Processing(3) RNase D is an exonuclease that trims in a 3' to 5' direction to generate the 3' endor the mature tRNA.Figure 3.3.3. The ends of tRNA in E. coli are produced by the action of three nucleases that cleavethe precursor to tRNA. A schematic of the pre-tRNA is shown at the top, with RNA extendingfrom the 5’ and 3’ ends of the RNA that will become the mature tRNA (shown as a cloverleaf).The site of cleavage is indicated by the short vertical arrows above the lines denoting RNA, andthey are labeled with the name of the enzyme cutting at that site. The enzymes catalyzing eachreaction are listed above or adjacent to the reaction arrows.b. The catalytic activity of RNase P is in the RNA component(1) RNAse P is composed of a 375 nt RNA and a 20 kDa protein.(2) The catalytic activity is in the RNA. The protein is thought to aid in the reaction,but is not required for catalysis. All enzymes are not proteins!(3) This was one of the first instances discovered of catalytic RNA, and SidneyAltman shared the Nobel Prize for this.BMB 400 PART THREE - III = Chpt.12. RNA ProcessingFig. 3.3.4. RNase Pc. The enzyme tRNA nucleotidyl transferase adds CCA to the 3' ends of pre-tRNAs.(1) Virtually all tRNAs end in CCA, forms the amino acceptor stem.(2) For most prokaryotic tRNA genes, the CCA is encoded at the 3' end of thegene.(3) No known eukaryotic tRNA gene encodes the CCA, but rather it is addedposttranscriptionally by the enzyme tRNA nucleotidyl transferase. This enzymeis present in a wide variety of organisms, including bacteria, in the latter casepresumably to add CCA to damaged tRNAs.C. Modifications at the 5' and 3' ends of mRNAAs discussed previously, eukaryotic mRNAs are capped at their 5' end and polyadenylated attheir 3' end. In vitro assays for these reactions have been developed, and several of theenzymatic activities have been identified. These will be reviewed in this section.Polyadenylation is not limited to eukaryotes. Several mRNAs in E. coli arepolyadenylated as well. This is a fairly new area of study.BMB 400 PART THREE - III = Chpt.12. RNA ProcessingFig. 3.3.5. mRNAs can be modified on the 5’ and 3’ ends.1. Modification at the 5' end: cap structurea. The "cap" is a methylated 5'-GMP that is linked via its 5' phosphate to theβ-phosphoryl of the initiating nucleotide (usually A). See Fig. 3.3.6.b. Capping occurs shortly after transcription has begun.c. It occurs in a series of enzymatic steps (Fig. 3.3.7).(1) Remove the γ-phosphoryl of the initiating nucleotide (RNA triphosphatase)(2) Link a GMP to the β-phosphoryl of the initiating nucleotide (mRNAguanylyl transferase). The GMP is derived from GTP, and is linked byits 5' phosphate to the 5' diphosphate of the initiating nucleotide.Pyrophosphate is released.(3) The N-7 of the cap GMP is methylated (methyl transferase), donor isS-adenosyl methionine.(4) Subsequent methylations occur on the 2' OH of the first two nucleotidesof the mRNA.d. Capping has been implicated in having a role in efficiency of translation andin mRNA stability.BMB 400 PART THREE - III = Chpt.12. RNA ProcessingFig. 3.3.6. Structure of the 5’ cap on eukaryotic mRNAs.Figure 3.3.7. Stepwise synthesis of the 5’ cap.BMB 400 PART THREE - III = Chpt.12. RNA Processing2. Several proteins are required for cleavage and polyadenylation at the 3' end.CPSF is a tetrameric specificity factor; it recognizes and binds to the AAUAAApolyadenylation signal.CFI and CFII are cleavage factors.PAP is the polyA polymerase.CFI, CFII and PAP form a complex that binds to the nascent RNA at the cleavage site,directed by the CPSF specificity factor.CstF is an additional protein implicated in this process in vitro, but its precise functionis currently unknown.Fig. 3.3.8BMB 400 PART THREE - III = Chpt.12. RNA ProcessingD. Multiple mechanisms are used for splicing different types of introns.1. Different types of intronsa. pre-tRNAb. group I, group II: Introns in fungal mitochondrial genes and in plastid (chloroplast)genes have been grouped into 2 different groups based on different consensussequences found in the introns. As we will see below, the group II introns have amechanism for splicing that