Prokaryotic Molecular Biology (1-3) Flashcards

Question

What is transposition?

Answer 1

Inversion sequences → many DNA sequences which don't transpose but can alter their orientation with a DNA molecule, inversions sequences control gene expression in some organisms e.g. phase variation in Salmonella spp. → most can produce 2 different types of flagellum phase 1 and 2 H antigens → growing phase 1 cells will produce some phase 2 cells and vice versa → phase is determined by the orientations of the *hin* (H-inversion) region

Answer 2

DNA in the cell is continuously exposed to damage → damage is any change from the normal nucleotide sequence and supercoiled double helical state → causes: physical and chemical agents in the environment e.g. UV light, free radicals produced during metabolism, errors in DNA replication

Answer 3

Single base changes → produces mutations but have no effect on physical process of transcription or replication, replication errors due to keto-enol type tautomerisation, deamination of cytosine to uracil, incorporation of U rather than T during replication, chemical modification of bases Structural distortions → may impede transcription and/or replication, single strand breaks, covalent modification of bases e.g. alkylation, removal of a base, inter strand and intrastrand covalent bonds

Answer 4

Standard base tautomers (structural isomers - readily interconvert) → standard base pairing arrangements - thymine (keto) adenine (amino), cytosine (amino) guanine (keto) → anomalous base pairing arrangements - thymine (enol) guanine (keto), cytosine (imino) adenine (amino)

Answer 5

Thymine dimer formation caused by UV light → two adjacent thymines on the same strand become covalently linked in a cyclobutane structure or a (6-4) photoproduct

Answer 6

Cells have systems which recognise mismatches and structural distortions in DNA and resolve these with a range of repair processes Direct repair → reversal or simple removal of the damage Mismatch repair → detection and repair of mismatched bases Excision repair → recognition of the damage followed by excision of a patch of DNA and its replacement by undamaged DNA Tolerance systems → allow DNA replication to process through damages regions of DNA Retrieval systems → recombinational processes to repair damaged DNA

Answer 7

Photolyase → photo reactivation is an example of direct repair → this process repairs any UV-induced intrastrand pyrimidine dimer (almost always thymine dimers) → the enzyme deoxyribopyrimidine photolysis binds specifically to pyrimidine (thymine) dimers in the dark → photolyase contains two chromophore that absorb light energy in the range 300-600nm - absorbed energy is used to split cyclobutane structures

Answer 8

Uracil DNA glycosylase (UDG) → uracil is occasionally incorporated into DNA instead of thymine → UDG removes uracil base form the nucleotide making an AP site (apurinic or apyrimidinic) → AP endonuclease makes a break in the phosophodiester backbone adjacent (5') to the AP site → DNA polymerase I binds to the break and lays down new DNA and the gap is sealed by DNA ligase The *mut* system → MutS recognises mismatched and short insertion/deletions (indels) on semi-methylated DNA and binds to them → MutL binds and stabilises the complex → the MutS-MutL complex activates MutH → MutH locates a nearby methyl group and nicks the newly synthesised strand opposite the methyl group → MutU (helicase II) unwinds the DNA from the nick in the direction of the mismatch → DNA PolI degrades and replaces the unwound DNA and DNA ligase seals the single strand break

Answer 9

In E. coli there are 3 excision repair modes: → very short patch (deals with mismatches between bases) → short patch ~20 nucleotides → long patch 1,500-10,000 bps Both short and long patch repair utilise the repair endonuclease → encoded by the uvrA, uvrB and uvrC genes Enzyme uvrABC binds to damaged regions makes and incision on both sides of the damage UvrD (alias MutU DNA helicase II) separates strands As in mismatch repair DNA polI replaces the DNA ligase fills the gap Short patch repair accounts for 99% of bulky lesions repair events Long patch repair is an inducible activity

Answer 10

Inducible error-prone repair Low-fidelity DNA polymerases (translation synthesis polymerases (TPSs)) can synthesise DNA past damaged bases → not efficient at replicating undamaged DNA accurately - most lacking proof-reading ability Two in E. coli: polymerases IV and V and 5 in human cells Almost all are members of new DNA pol family and Y-family In some circumstances make many errors so can generate mutations Each TSP appears to have a different substrate specificity → human polymerase eta can bypass the major UV photoproduct very efficiently, usually inserting the correct nucleotides - less efficient with most other types of damage → defective in the variant form of a highly skin-cancer-prone genetic disorder, xeroderma pigmentosum, - so eta helps prevent UV-induces mutations in cancer

Answer 11

Daughter strand gap repair → does not actually repair damage → permits replication to occur successfully → relies on other repair processes such as excision repair to repair the damage afterwards The SOS response in E. coli → if it suffers severe DNA damage it activates the expression of a large number of diverse unlinked genes involved in DNA repair, error prone replication etc. → all genes and operons under SOS control are subject to repression by the LexA protein - 2 domains: dimerisation and a DNA-binding domain → conserved binding site "LexA box' located within th w-promoter of genes regulated by LexA → binding of LexA to the LexA boxes represses expression of SOS operons RecA proteins is involved in inducing the SOS response → responds to DNA damage changes conformation which activates it - inhibition of the 3'-5' editing in DNA Pol III allowing error-prone DNA replication, interacts with LexA which autoclaves becomes inactive leading to the SOS repose - sulA expression inhibits cell division → one DNA damage is repaired RecA* converts back SOS réponse allows the cell to survive severe DNA damage by allowing DNA replication but at the expense of fidelity - last ditch effort by the cell to replicate with severe DNA damage

Answer 12

Induction - switching on of genes when they are required Repression - switching off of genes when they are not required Regulatory proteins interact with DNA → repressors - prevent transcription when bound to DNA → activators - activate transcription when bound to DNA converted between inactive and active states by effectors → inducers - activate activators or inactivate repressors - switch genes on → co-repressors - activate repressors or inactive activators - switch genes off

Answer 13

Genes associated with a particular physiological function may not be in just one operon → these operons may be controlled by a single regulatory protein - together called a regulon e.g. phosphate or PHO regulon >80 genes in many operons controlled by one regulatory protein

Answer 14

*E. coli* prefers to use glucose as its carbon source → if both glucose and an alternative carbon source is available the glucose is used first - diauxic growth → has diauxic lag when switching between carbon sources

Answer 15

Glucose represses the synthesis of enzymes that metabolise less preferred carbon sources - catabolite repression → if lactose not available and glucose is *E. coli* will have a small amount of enzymes that metabolise lactose → when the cells runs out of glucose there is rapid induction of the enzymes for lactose metabolism When glucose absent activates adenylate cyclase which turns ATP into cAMP induces CAP binding → RNA polymerase binds p → lactose present activates allolactose which binds o → stimulates expression of lacZ lacY and lacA On the lac operon lacI → inhibits mRNA production for the proteins encoded by the lac operon - encodes a repressor protein → allolactose binds the repressor, stops repression - allows transcription of the lac genes

Answer 16

Trans-acting elements → some genes encoded for diffusible products that regulated gene expression on both DNA molecules Cis-acting elements → other genes dit not produce a produce but regulated genes on the DNA molecule they were encoded upon

Answer 17

In E.coli lac operon → both normal and Lac+ inducible operons repressed → allolcatose induces expression on both molecules → chromosome produces a functional LacZ → F' plasmid produces a functional LacY → these complement the two mutated genes so phenotype is normal