BMB 462 Lecture 31 Outline of Last Lecture I. Rho-dependent terminationII. Differences between eukaryotic and bacterial transcriptionIII. RNA Polymerase IIV. RNA Polymerase IIV. RNA Polymerase IIIVI. Transcription inhibitorsOutline of Current Lecture I. Introduction to RNA processingII. 5’ cappingIII. Splicing of pre-mRNAIV. Transesterification of intronsV. Self-splicing intronsa. Group Ib. Group IIVI. Spliceosomal intronsCurrent LectureConcepts to remembers from previous courses/lectures:-I. Introduction to RNA processinga. Primary transcripts (i.e. pre-mRNAs) are converted to functional RNAs. Particularly in eukaryotes, RNAs have to be processed before they're functional.b. RNA is not typically processed in bacteria due to the fact that transcription and translation are coupled.II. 5’ cappinga. 5' capping of the mRNA occurs during synthesis as it emerges from RNA polymerase IIb. The pre-mRNA is composed of exons and introns. The exons will remain after processing, and the introns will be removed via splicing of the mRNAi. Splicing also occurs while the transcript is being made.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.c. To finish the processing, the 3' untranslated region (UTR) is cleaved. Polyadenylation adds a poly(A) tail to the 3' end. There are typically 80-250 ‘A’s inthe tail. The poly(A) tail is a message that marks the mRNA as complete and ready to move from the nucleus out into the cell.d. The introns leave as lariat structures after splicing.e. The 5' cap protects the mRNA from degradation due to exoribonuclease activity. The cap is also necessary for translation because it is recognized by ribosomes.f. 5' capping involves putting a 7-methyl-guanosine on the 5' end of the mRNA in an unusual 5'-5' triphosphate linkage. This unusual structure is what helps protect it from RNasesg. The first nucleotide of the RNA can also on occasion be methylated in the 2' position. Sometimes even the 2nd nucleotide can be methylated at the 2' positionh. RNA polymerases start synthesis de novo (they do not need a primer) so the first nucleotide still has its triphosphate attached to the 5' end. The first step is then that phosphohydrolase removes the gamma phosphate. Then, guanylyltransferase attaches a guanine from GTP by its α phosphate to the beta phosphate of the nucleotide. The leaving group of this reaction is a pyrophosphate from GTP.i. Next guanine-7-methyltransferase adds a 7-methyl group to the newly added guanine. It uses S-adenosyl-methionine to do soi. The 7-methyl-guanine has now been added in a 5' to 5' linkage.j. Next, a 2'-O-methyltransferase can transfer a methyl group to the 2' position of the first nucleotide in the RNAk. 5' Capping in the Context of transcription:i. The phosphorylated CTD of the RNA polymerase II begins to leave the promoter region, having initiated a chainii. The cap-synthesizing complex is associated with the phosphorylated CTD. As the 5' end of the RNA emerges out of RNA polymerase II, it interacts with the cap-synthesizing complex; the cap is made once the RNA is 20-30nucleotides long (that's when it's long enough to have come far enough out of the RNA polymerase to be recognized by the cap-synthesizing complex).iii. Once the cap has been made, the cap-synthesizing complex dissociates, the cap-binding complex (CBC) associates to the CTD. This keeps the cap tethered to the CTD during elongation, ensuring the RNA remains in closeproximity during its synthesis. This is important because the sNRPs involved in splicing also associate with the CTD, and tethering keeps the RNA in place for further processing. The cap protects the RNA fromRNases and is used for recognition by elongation and other processing factors.III. Splicing of pre-mRNAa. There are multiple types of introns; introns were not discovered until 1977i. Most genes in (higher order) vertebrates have introns. The genes for histones, though, are the exception and do not. This is because the cell needs to make a lot of histones, so doesn't spend time splicing the mRNAs needed for histone production.b. The human genome is composed of ~1.5% exons and 28.5% introns. The rest of the genome is composed of repetitive sequences, jump DNA (though this idea is falling into disfavour as scientists discover more about noncoding RNAs)c. Most genes in yeast - which is a primitive, microbial eukaryote - lack introns, and only a very few bacterial genes have introns.i. This is so that less time and energy is spent on RNA processing, so the cells can grow and replicate faster.d. Introns range from ~50-20,000 nucleotides long while exons are typically shorter at 100-1000 nucleotides.IV. Transesterification of intronsa. Transesterification reactions are involved in making 3 of the 4 intron types.i. A phosphodiester bond is broken and exchanged, so the cell doesn't needenergy from ATP in order to accomplish splicing. Instead, the reaction components are held close together so that the reaction is catalyzed. Enzymes and ribosomes hold things in proximity so that a chemical reaction can occur.b. For example, the 1st step in Group 1 intron splicing: The phosphodiester bond between the last nucleotides of the intron and exon is broken. A new bond is formed between the 5' phosphate of the intron nucleotide and the 3' hydroxyl group of a guanosine.c. This exchange of phosphodiester bonds is accomplished without the input of energy due to bringing the guanosine in very close proximity to the bond between the last nucleotide of the exon and the first nucleotide of the intron (the splice site).V. Self-splicing intronsa. Self-splicing introns do not use proteins; the process is completed by ribozymes.i. Group I introns use guanosine, GMP, GDP, or GTP (a guanosine or a guanosine-containing nucleotide).ii. Group II use the 2' hydroxyl of an A (aka the Critical A) in the intron.1. The group II mechanism is very similar to the spliceosomal mechanism that relies on a combination of small RNAs and proteins called sNRPs.b. Group I self-splicing introns were discovered in 1982 by Thomas Cechi. He was given the Nobel Prize for his work; he was the fist to describe a ribozyme. (No one had the concept of a catalytic RNA up until that point).1. The Group 1 self-splicing introns were discovered in tetrahymena, a small, single celled eukaryotic organism; the intron was found in a rRNA that relies on a
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