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Molekularbiologie (Subject) / transkription (Lesson)

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  • 109. What are the major two differences between RNA and DNa? ·         RNA has Uracil instead of Thymin ·         RNA has Ribose instead of Desoxyribose
  • 110. Name the three major phases in transcription. ·        Initiation ·        Elongation ·         Termination
  • 111. Name the three major eukaryotic RNA Polymerase and their major role! ·         RNA Polymerase 1: large ribosomal RNA ·         RNA Polymerase 2: mRNA ·         RNA Polymerase 3: tRNA and 5s rRNA
  • 112. What is special for plant RNA Polymerase? Have an additional RNA 4 Polymerase, which transcribes rRNA (regulatory RNA)
  • 113. When it comes to how much work RNA polymerases have, how would you rate the transcription of RNA Polymerase I+III 0/4< x <1/4< x <2/4< x <3/4< x <4/4 ? RNA Polymerase 1 and 3 are responsible for 80 % of transcription.
  • 114. What can happen to the C-terminal domain of eukaryotic RNA polymerase II?                 It can become phosphorylated.
  • 115. What does the bacterial sigma factor do? ·         RNA core polymerase cannot find transcription start by itself. ·         Another protein is needed to direct the RNA core Polymerase to the promoter. ·         The core bacterial enzyme needs an additional subunit the sigma factor, which contacts the promoter. ·         Sigma factor plus enzyme = Bacterial Holoenzyme!
  • 116. What are the major two bacterial promoter elements (that are close to the TSS) (for sigma70) (hint name them by position)? -35 and -10 element. These are roughly 35 and 10 bases upstream of the transcription start 
  • 117. In which bacterial promoters do you find the UP element? (somewhat active, medium active, very active)? very active
  • 118. Anti sigma factors? Proteins, which binds to sigma factors and inhibit their function!
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  • 119. In S. typhimurium flagellum assembly an anti sigma factor is involved, can you say how this factor FlgM is later loosing its influence. It is exported through the incomplete flagellum apparatus.
  • 120. Eukaryotic promoters have a box DNA Plymerase need to initiate transcription. TATA box
  • 121. Which protein do all three eukaryotic RNA polymerases need for establishing the pre-initiaion complex? TATA binding protein TBP
  • 122. When it comes to transcription what is the hallmark of a closed complex? RNA polymerase is in position and the DNA is not yet opened, then the complex of polymerase and promoter is called closed complex,
  • 123. When it comes to transcription what is the hallmark of an open complex? RNA polymerase has opened up the transcription bubble. Open complex wenn Polymerase bound to open replication bubble!
  • 124. Interestingly, when RNA polymerase starts making RNA, a strange phenomena occurs resulting in short pieces of RNA, explain this? RNA polymerase is not immediately working fully instead it enters cycles of abortive initiation resulting in short pieces of RNA being released. This is likely due to a loop of eukaryotic TFIIB and bacterial sigma factors extending into the polymerase
  • 125. What is promoter clearance? When the loop of TFIIB/sigma inserted into the RNA polymerase is displaced and the polymerase breaks away from the promoter.
  • 126. What happens to the CTD when eukaryotic RNA polymerase is converted into the elongating complex after promoter clearance? It becomes phosphorylated
  • 127. Describe a simple! experiment to show that abortive initiation takes place in vitro. Abortive initiation was discovered in E. coli using the lacUV5 promoter. DNA containing this promoter was incubated with radioactive nucleotides and RNA polymerase Holoenzyme. Run products on gel and visualize with Xray film. 
  • 128. To show that abortive initiation takes place in vivo special kinds of nucleic acids were used, name them and indicate why these were used? locked nucleic acids (these are locked in a conformation more favourable for hybridization)
  • 129. What is the approximate speed of RNA polymerase 0<x <10 x <100< x <1000< x <10000< x <100000 nucleotides per second? 10< x <100 (20-50 nt/s)
  • 130. What is promoter proximal pausing? When there is some pausing after only 35-50 bp have been synthesized by RNA polymerase. 
  • 131. What are potential reasons for transcriptional pausing? ·         Short complementary regions in the nascent transcript forming hairpins or ·         weak DNA-RNA hybrids
  • 132. Which factors can relieve transcriptional pausing?               Elongation factors
  • 133. Explain how transcriptional pausing may be relieved? RNA polymerases reverses direction, most recently made RNA separates off from DNA, this protruding end can be chopped off.
  • 134. What is one of the first maturation steps that occurs to eukaryotic mRNA?                         This is often capping, happening while RNA is still being made. 
  • 135. What happens after capping to the eukaryotic RNA polymerase II CTD? It is phosphorylated at additional serines.
  • 136. What is the role of the 5' cap for the mRNA? ·         Capping the 5’ end protects the mRNAs from nuclease degradation, ·         helps in efficient elongation and termination of the transcript, ·         in mRNA processing and export from the nucleus ·         directing translation
  • 137. What is the minimal structure incorporated into a cap? 7-methylguanine is lined via a 5'-5' triphosphate to the 5' end of the RNA
  • 138. Describe schematically how capping proceeds? The 5’ cap is added in three stages: 1. the 5’ phosphate is removed 2. a guanyl transferase attaches a guanosine monophosphate (GMP) to the end in a 5’ -5’ triphosphate linkage  3. the guanine is methylated by a guanine-7-methyl transferase 
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  • 139. What takes care of the nucleosome problem during transcription?   Eukaryotes use histone chaperones to remove nucleosomes ahead of RNA polymerase, and reassemble them behind the polymerase.
  • 140. What topological problem can transcription cause? This leads to changes in supercoiling – there is an increase in positive supercoiling ahead of the polymerase and an increase in negative supercoiling behind the polymerase
  • 141. What is a (bacterial) intrinsic terminator? A site where bacterial RNA polymerase terminates transcription without any additional factors.
  • 142. What are the two main features of a bacterial terminator? ·         inverted repeat that forms a stem loop in the RNA ·         a polyA (DNA) region, that leads to less stable AU base pairs
  • 143. What is the bacterial Rho protein (in the field transcription)? ·         An enzymatic terminator
  • 144. What is the difference between intrinsic and Rho dependent genes in terms of termination? The former make a hairpin and have a poly A region the latter (Rgho) don't
  • 145. Which eukaryotic RNA polymerase recognizes intrinsic terminators? RNA Polymerase III
  • 146. When you compare bacterial intrinsic terminators to eu. RNA polymerase III terminators what does the latter not need? eu. RNA Pol III doesn't need the RNA hairpin 6a/46
  • 147. What is eu. RNA polymerase II termination coupled to? It is coupled to 3' end processing
  • 148. What happens to most eukaryotic 3' mRNAs? They get polyadenylated
  • 149. Is the polyA tail of eu. mRNA added to the last based made by RNA polymerase II? No, the nascent transcript is cleaved first and then the polyA tail is added.
  • 150. Give the names of the two major model for eu. RNA polymerase II termination? torpedo and allosteric model
  • 151. What must a RNA transcription regulatory protein do? It must specifically recognize its right regulatory sequence e.g. through a protein domain
  • 152. Why can it be favourable to contact the major grove of DNA in terms of specificity? In the major grove all bases can potentially be distinguished this is not necessarily the case for the minor grove.
  • 153. Which kind of amino acids would you expect to find in proteins that contact DNA and why? positively charged amino acids, as the DNA is negatively charged
  • 154. Give two examples of domains or motifs found in transcription factors? helix-turn-helix, homeodomain, zinc finger....
  • 155. Describe the procaryotic trp repressor system? • The Trp repressor is a helix-turn-helix that responds to the level of cellular tryptophan   •The genes needed to synthesis tryptophan are located, regulated and transcribed together. This configuration is called an operon.  • When tryptophan levels are low => genes are transcribed, to make tryptophan  • When tryptophan levels are high => tryptophan repressor binds to the Trp operator and blocks RNA polymerase from binding  • The Trp repressor can only bind to DNA when it is itself bound by tryptophan, which only happens when tryptophan levels are high  
  • 156. The lac system depends on the input of both glucose and lactose availability. Name the promoter states (off/weakly on/strongly on) in dependence of glucose and lactose concentration? low glucose, lactose available -> strongly on     low glucose, lactose not available -> off     high glucose, lactose available -> weakly on     high glucose, lactose not available -> off
  • 157. A colleague of yours proposes that bacteria don't phosphorylate proteins. Can you prove him wrong by naming a signal transduction pathway? The two component signal transduction pathway features a histidine kinase.
  • 158. Can transcriptional regulation only occur in the initiation phase? No it can also occur during elongation and termination

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