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• 29. Was ist transition und transversion? Transition: Purin vs. Purin, Pyrimidin vs. Pyrimidin Tranversion: Purin vs. Pyrimidin, vice versa
• 30. Name some sources which can lead to mutation? ·         Radiation (UV) ·         Internal cell metabolites ·         Tautomeric structures of DNa bases ·         External chemicals ·         Deamination
• 31. What is a simple mechanism to repair UV induced tymine dimerization? (direct reversal) Direct reversal of the damage by photolyases. For example they reverse pyrimidin dimerization.
• 32. Alkylation repair. What is special about it? (direct reversal) Alkylation on guanine or the DNA backbone is repaired by alkyltransferase. Once bound to an alkyl group Ada is inactivated. Methyl Ada stimulates more Ada production and also stimulates AlkA production (another repair protein). Ada transfers a methyl group from the damaged alkylated DNA to its active sites. Methylated Ada activates expression of its own gene and a glycosylase gene. 
• 33. Mismatch repair? Mis match repair fixes mistakes made during replication, using the other strand as a template. The mismatch is recognized by MutS protein the new strand by MutH (new DNA is not methylated).  MutSLH complex assembles and MutH nicks the new strand. The nicked strand is digested by an exonuclease and DNA is resynthesized. Mismatch repair can detect and repair hairpins – the newly synthesized DNA is degraded, the hairpin unfolds and the new strand can be re-made. Defects in mismatch repair lead to increased rates of cancer and spontaneous mutation (this is a mutator phenotype). 
• 34. Base Excision repair? When it is used? Damaged bases are removed by glycosylases. AP endonucleases cleave the backbone and the gap is filled by repair polymerases. AP endouncleases also become active if thebase has been lost for any other reason. 
• 35. Nucleotide excision repair. It is used for bulky lesions, which distort the DNA helix and 10-30 bases are excised and repaced. In bacteria, UvrA and UvrB scan DNA for distorted regions, UvrB (a helicase) unwinds the damaged region. UvrC is recruited and nicks the damaged DNA and damaged DNA is then removed by UvrD. DNA is resynthesized from the undamaged strand. 
• 36. TLS Polymerase? Damaged DNA may fail to be repaired before it meets a replication fork, and can lead to stalled replication forks. After replication forks stall on damaged DNA, translesion polymerases (TLS) can take over synthesis. TLS polymerases can work in a non-template fashion. After TLS polymerases finish, the normal polymerases take over once again. Because of their low fidelity, TLS polymerases must only be recruited when necessary. TLS polymerase active sites are more open and flexible than replicative polymerases. This flexibility allows then to bypass problematic DNA and continue synthesis, albeit with lower fidelity. 
• 37. Why are TLS polymerase a last resort? The TLs polymerase are error prone and just ensure that DNa can be replicated, this often leads to mutations. It can safeguard replication though. 
• 38. LEXA/RECA system. ·         damage response ·          replication fork stallsà single-stranded DNA is exposed – RecA binds ·          Bound RecA forms a filament on single-stranded DNA, and stimulates cleavage of LexA. Cleaved LexA can’t bind DNA, SOS genes are transcribed. SOS repair proteins fix the damaged DNA. RecA activity decreases, and LexA rebinds DNA to turn off SOS gene transcription. LexA also inhibits cell cycle progression, to gain time for damage repair. 
• 39. Where do ATM and ATR play a role? They play a role in single and double stranded DNA breaks in eukatiots. Sensors recruit the Regulator kinases (ATM and ATR), which mediate the damage response. 
• 40. Effects of atr activation? o   Cell cycle control o   Replication fork stabilization o   Replication origin control
• Which kind of processing steps can eukaryotic RNA encounter? Cleaving, splicing, polyAdenylation, Capping, Editing
• Do all RNA´s have the same half-life? No, it can range between 1 min to an hour in E. coli and 20 min to >24 hrs.
• What effects RNA stability? ·         3´Poly-(A)-tail ·         Stem loop structures at 5´ and 3´ ·         5´and 3´structures
• Explain RNA degradation. Bacterial:    o   Degradation is initiated by endonuclease, usally RNAs E o   5 triphosphate inhibits RNase E activity. This is converted to a monophosphate by RPPH o   Products of first RNase digestion are converted by 3´to 5´ exonucleases o   RNase E is part of the degradosome complex. The complex also has an RNA helicase and a 3′ to 5′ exonuclease eukaryotic: §  Endonuclease activity can also initiatiate degradation in eukaryotes §  • 3′ poly(A) tails hinder rather than aid degradation in eukaryotes – the first §  step in degradation is often tail shortening by a deadenylase §  • After this, decapping enzymes remove the 5′ cap and 5′ to 3′ exonucleases §  degrade the RNA, or the exosome catalyzes exonuclease activity in the 3′ to §  5′ direction
• 178. Which nucleotide is used by EF-TU/Ts? o   GTP
• 179. Which trick to translation factors often use to access the ribosome interface? They  can mimic the RNA. 
• 180. Apart from a start codon which other sequence do you need in bacteria to start translation? o   Shine Delargano sequence: Initian codon, which have theses sequence. A polypurine tract, which is 6-8 bases upstream of the initiator AUG.
• 181. Kozak sequence? A sequence with A/GXXAUGG, which helps to recognise the right AUG in eukatiots.
• 182. Closed loop complex in translation? ·         eukaryotic mRNA 5' cap + 3' polyA tail  involved in initiation ·         àpreparing the mRNA to be scanned by the ribosome. The ·          cap is bound by eIF4E and the tail by PABP ·         These interact with each other via a complex of other factors. ·         This forms a closed loop complex ·         Function:  quality control to weed out unfinished or damaged mRNAs.
• 183. Is stop recognition sloppy? No, it is highly accurate. Premature peptide release is very rarely (1/100 000 events)
• 184. What is tmRNA used for, where it is used and how? ·         Quality control in bacterial translation to discriminate between full length and truncated tmRNA ·         A tmRNA (tRNA-like and mRNA-like) acts as a tRNA and adds an alanine to the peptide, then act as an mRNA with a short ORF and termination codon. ·         The short “mRNA” from the tmRNA is then translated into an 11 amino acid “tag”, attached to the previously translated peptide. ·         The “tag” signals the whole peptide for degradation.
• 185. Explain the 21st amino acid and its protein incorporation. o   21st AA, similar to cysteine has a selenium instead of a  sulphur. o   It is operated into catalytic sites in several enzymes, where it can act as an strong reducing agent. o   Selenocystein
• 186. Is Pyrrolysine a proteinogenic amino acid? A 22nd amino acid is a modified lysine and is found in methyltransferase genes at catalytic sites. It is incorporated at the UGA stop codon in a similar way to selenocysteine. It is a proteinogenic amino acid.
• 187. What is the magic spot? Translation cannot proceed with an uncharged tRNA in the A site but this binding also recruits RelA. RelA synthesizes lots of (p)ppGpp (a pentaphosphate guanine nucleotide called “magic spot”). This modulates transcription and induces a stress responses to replenish amino acids. This is called the “stringent response”
• 188. Give an example for the less common 5' UTR regulation in eukaryotes? 5' UTR regulation is used for iron regulation. Too much free iron is toxic. So iron is tightly bound to ferritin. The higher the iron concentration the more ferritin is needed. The 5' UTR of ferritin mRNA has Iron Response Elements (IREs) that bind to Iron Regulatory Proteins (IRPs). If Iron is scarce IRPs bind to IREs and stop translation by preventing ribosomes from accessing AUG. If there is lots of iron, then iron binds to IRPs which then cannot bind IREs and translation proceeds.
• 189. How is it achieved that in X. laevis oocytes mRNA is initially not translated? In X. laevis oocytes maternally-derived mRNAs are not initially translated. They are dormant or translationally repressed. Translation depends on the 3' UTR polyA tail length which is initially short. The 3' UTR cytoplasmic polyadenylation element (CPE) is bound by CPEB. CPEB sequesters eIF4E via other proteins and this stops formation of the closed loop required for translation initiation. Dormant mRNAs are activated by phosphorylation of CPEB which eventually leads to extension of the polyA tail nd dislodging of the inhibitory complex. EIF4E can then bind to eIF4G, form the closed loop complex and initiate translatio
• 190. How is splicing coupled to NMD? Premature stop codons (PTCs) can occur in an mRNA by mistake. Most are recognized and degraded by nonsense mediated decay (NMD). PTCs (premature stop codons) must be recognized as faulty. After splicing the exon junction complex (EJC) remains at the exon-exon junction. Therefore EJCs are not found after a real stop codon but will occur after most PTCs. NMD is triggered by ribosomes encountering a stop codon upstream of an EJC.
• Was ist so beeindruckend an RNA und warum träumen manche über eine RNA Welt? ·         RNA speichert sehr viele genetische Informationen ·         RNA kann als Enzym agieren
• Warum muss DNA gepackt werden? DNA sehr langes Molekül und muss in die Zelle bzw. in den Zellkern reinpassen
• Haben Bakterien Histone? Nein sie haben ähnliche positiv geladene Proteine den IHF (Integration host factor). Dieser bindet an die DNa und biegt sie. Das biegen induziert das supercoiling.
• Wie heißen die 4 Histone, die einen core particel ausmachen? H2A, H2B, H3, H4
• Wo spielt H1 eine Rolle? The higher packaged 30 nm fiber requieres the H1. H1 is a linker histone that binds to the linker DNA in between successive nucleosomes, helping compaction.
• How long is a stretch around the nucleosome? About 146 base pairs.
• Wie kann man die Länge der DNA um einen Nucleosom bestimmen? Die DNa, die nicht um den Histon ist wird mit der microccocal nuclease behandelt. Die DNa um den Histon herum wird geschützt und nicht von dem Enzym angegriffen. Die DNa, die übrigt bleibt wird durch die Gelelektrophorese nach Größe getrennt.
• If you digest chromosomal DNA with micrococal DNA and run them on a gel you get shortish fragments of around 170-200bp but also longer and much longer bands, where are these coming from? Shorter fragments arise from degradation. ·         Longer fragments are cut in the middle. ·         Fragments with a multiple number arise from incomplete digestion.
• Welche Histonmodifikationen gibt es? ·        Acetylation (Ac) ·        Ubiquitination (Ub) ·        Methylation (Me) ·        Phosphorylation (P) ·        Sumoylation (Su)
• Was bedeutet HAT? Histoneacetyltransferase
• Was machen PRC1 und PRC2 und wie machen sie es? PRC=Polycomb repressive complex (protein) ·         PRC2 silence gene via histone modification. This complex has histone methyltransferase activity and primarily trimethylates histone H3 on lysine 27 ·         PRC1 is required for stabilizing this silencing and underlies cellular memory of silenced region after cellular differentiation
• Was ist LHP1? PRC1 like protein in plants. It stabilize the silencing state. 
• Was ist H3K27me3? Mit was wird die Histonemodifikation assoziiert? Histone 3 Lysine 27 triple methylated It is associated with genes. 
• Welche chem. Modifikationen von Histonen gibt es? o   Methylierung von cytosinen! o   Mathylation of Adenin
• Manchmal muss gepackte DNa erreicht werden. Wie geschicht dies? Nucleosome position can be changed to allow proteins to acess the DNa.  ATP-dependent nucleosome remodeling complexes increase accessibility of DNA by: - Sliding the histone octamer along the DNA, or - Removing the histone octamer and transferring it elsewhere, or - Introducing loops into the DNA wrapped round a histone core Several classes of nucleosome remodeling complexes exist, with different subunit compositions, such as: - - SWI/SNF: disrupts nucleosome positioning - ACF (ISWI family): positions nucleosomes during chromatin assembl
• Was sind Bromo und Chromodomain? An welche Strukturen binden diese? These are subunits of remodelling complexes. Structural domain of about 40-50 aa. Proteins. ·         Bromodomain, which binds to acetylated lysines ·         Chromodomain which binds to methylated Lysines
• 17. Why is DNA methylation risky? Because DNa can be permanently altered. Methylated Cytosines are less stable, so more likely to undergo deamination, which change the base to Thymin.
• What is DNA Metyhlation used for in bacteria? In bacteria it is used for distinguishing between newly synthesized and old DNa in repair process. Im mismatch repair, the methylated strand is read. This allows the repair machinery to detect the parental strand.  Secondly it allows bacteria to distinguish between genomic DNa and invading bacteriophage DNa (Restriction enzymes cut the DNa. Bacteria defending their own DNa by methylating the recognition sites).
• What are restriction enzymes?  Enzymes recognizing specific sites in the DNA and cutting the DNA.
• Do all eukaryotes use the same DNA methylation sites? No animals use mostly CG sites, Plants CG, CHG, CHH
• What is the advantage of CG methylation sites? CG methylation sites are symmetric and easier to maintain! Asymmetric methylation has a more complex maintain status. 

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