Biolchem 415 1st EditionExam # 4 Study Guide Lectures: 33 - 37This exam is cumulative. Review study guides 1-3 along with this study guide. Lecture 33 (April 10)What is a gene and how is its expression (eukaryotes) regulated? Why is it regulated?A gene is a region of DNA that is transcribed to produce a functional RNA molecule. Genes can encode an RNA molecule that performs biochemical functions such as rRNA, tRNA, or other non-protein encoding RNAs. Gene expression is regulated in order to generate different cell types. This is why genes have specificity. Gene expression is regulated in three ways. Regulation of transcription is very complex (having three different types of RNA polymerase). How RNA is processed is highly regulated, especially that of mRNA. Lastly the nuclear membrane regulated gene expression. This membrane separates the sites of RNA synthesis from that of protein synthesis with RNA transcription occurring in the nucleus and protein translation occurring in the cytoplasm. How many types of RNA polymerase do eukaryotes have? What are their differences? How doesα-amanitin (an RNA polymerase poison) affect each of them?There are three different types of eukaryotic RNA polymerase. Polymerase I is found in the nucleolus. It functions for cellular transcripts of 18S, 5.8S, and 28S rRNA. This polymerase is insensitive to the effects of α-amanitin. RNA polymerase II is found in the nucleoplasm and functions for cellular transcripts of mRNA precursors and snRNA. This polymerase is strongly inhibited by α-amanitin, which binds near the active site of RNA polymerase and prevents the translocation along the DNA. RNA polymerase III is found in the nucleoplasm and functions for the cellular transcripts of tRNA and 5S rRNA. This polymerase is only inhibited by α-amanitin in high concentrations. What are some examples of non-coding RNAs? What are their functions?rRNA – protein synthesis (holds mRNA and tRNA in place during synthesis) | tRNA – protein synthesis (carries activated amino acids) | snRNA – components of RNA splicing machinery (produce mature RNA from precursors) | snoRNA – rRNA biogenesis and modification | miRNA –regulates use of mRNA (in association with argonaute) | siRNA – antiviral defense mRNA degradation (splicing) | piRNA – gene regulation | lncRNA – gene regulationExplain what type of promoters each polymerase has and what they transcribe.RNA polymerase II transcribes mRNA. There are TATA containing promoters and TATA-less promoters. TATA containing promoters contain a TATA box followed by an initiator element. TATA-less promoters have an initiator element followed by a downstream promoter element. RNA polymerase I transcribes rRNA and is located in the nucleolus. Its promoter consists of an upstream promoter element followed by a ribosomal initiator. RNA polymerase III transcribes tRNA and an rRNA. There are two types of promoters for polymerase III. One is an A block followed by a C block. This promoter transcribes for the 5S rRNA. The second type is an A block followed by a break followed by a B block. This promoter transcribes for tRNA. Describe transcription initiation by RNA polymerase II.Transcription initiation requires the transcription factors TFIIA, B, D, E, F, and H. TFIID is the first to bind to the TATA box. This is followed sequentially by TFIIA, TFIIB, TFIIF, RNA polymerase II, TFIIE, and TFIIH. TFIIH opens the double helix of the DNA. It also phosphorylates the carboxyl-terminal domain of RNA polymerase II, which allows the polymerase to leave the promoter and begin transcription. (This is the transition from initiation to elongation.)What are some ways that transcription is further regulated?DNA binding regulatory proteins such as activators (bind enhances are stimulate transcription) and repressors (reduce or prevent transcription of a gene). DNA looping can also regulate transcription. This looping allows enhancers to come into contact with promoters. Multiple regulators can work together. This is called combinatorial control. How does DNA packaging affect transcription? How can this be overcome?DNA packaging by chromatin can impede transcription. Coactivators can stimulate transcriptionby loosening the interaction between histones and DNA, making the DNA more accessbile to transcriptional machinery. Histone modification by methylation, phosphorylation or more can also result in a remodeling of chromatin resulting in DNA being more accessbile to transcriptional machinery. An example of this is histone acetylation. This is done by Histone AcetylTransferase enzymes (HATS). This acetylation reduces the histones’ affinity for DNA and generates a docking site for proteins with bromodomains (acetyl-lysine binding domains). This leads to the recruitment of additional components of transcriptional machinery and RNA polymerase. Lecture 34 (April 13)How is mature ribosomal RNA generated?rRNA is generated by the cleavage of a precursor RNA. The precursor RNAs are synthesized by RNA polymerase I. The precursor rRNA undergoes nucleotide modification due to smallnucleolar ribonucleoproteins (snoRNP). These add methyl groups to the sections of the precursorRNA that will become mature RNA. Lastly, RNase enzymes cleave apart the precursor RNA leaving the mature RNA intact. Explain how tRNA is processed.RNA polymerase III catalyzes the synthesis process of precursors to tRNA. First RNase P cuts off the leader sequence that exists at the 5’end of the RNA. A CCA-adding enzyme adds a CCA sequence to the 3’end. This is the site where activated amino acids will be attached. Lastly, the intron is removed and the tRNA assumes a form with the anticodon at a bottom loop of the RNA. tRNA serves as an adaptor meaning that it recognizes the mRNA code. Describe how mRNA is processed from precursor transcripts. mRNA precursors are synthesized by RNA polymerase II and then processed. The first step of thisprocessing is the addition of a 5’ cap. This cap is a methylated GTP added to the 5’end of the RNA through a 5’-5’ triphosphate linkage. This cap allows further processing of the mRNA, prevents degradation of the RNA, and enhances translation. Next is the addition of a 3’ poly-adenylate tail. This tail increases translation efficiency, stabilizes the RNA, and also prevent degradation. The poly(A) tail is also recognized by poyl(A) binding proteins (PABP) that promote translation. Lastly, introns are removed via splicing. The