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Flashcards by Anna Hogarth, updated more than 1 year ago
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Created by Anna Hogarth
almost 7 years ago
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| Question | Answer |
| What is the function of osteoblasts and osteoclasts? | 1) Secrete bone 2) Absorbe bone |
| What does the pancreas secrete? | Insulin and glucagon to regulate carbohydrate metabolism |
| Which cells have different DNA content? | 1) Gametes 2) Red blood cells 3) White blood cells - T cells and B cells have had specfic regions rearrange |
| What are 'housekeeping genes'? | Cells which are involved in regulating cell metabolism etc. |
| What governs the identity of a cell and determines its function? | The correct temporal and spatial expression of specific genes |
| What are the steps from DNA to active protein? | 1) DNA to primary transcript which is regulated by transcriptional control 2) Primary transcript (pre-mRNA) to mRNA by processing control 3) mRNA leaving to the cytoplasm is regulated by transport control 4) mRNA to active protein - translation control 5) Active protein to inactive protein is regulated by protein activity control, such as phosphorylation |
| What happens if mRNA is not translated into an active protein? Give an example of genes/proteins (2) which are particularly susceptible. | mRNA stability control - mRNA can be degraded. Fos and Jun are immediate early genes which are uniquely susceptible to mRNA stability - control phases of the cell cycle. |
| What % of the control of going from DNA to protein arises from transcriptional control? | ~95% |
| What does Polymerase I transcription make? What is this important for? | 1) rRNA 2) Structural and catalytic subunits of ribosomes |
| Polymerase II? | 1) mRNA - code for proteins 2) miRNA - regulate mRNA translation 3) snRNA - Bridge splicing events 4) snoRNA - Guide methylation on target RNAs 5) IncRNA - Chromatin binding and protein scaffolds |
| Polymerase III? | 1) tRNA - amino acid carriers in translation 2) miRNA (some) - regulates mRNA translation 3) snRNA - Bridge splicing events |
| What are the steps in mRNA synthesis? | 1) Initiation - the polymerase binds to the gene 2) Elongation - the polymerase transcribes the gene (polymerase travels along the gene and makes the RNA) 3) Termination - the polymerase stops transcribing the gene 4) Processing - the mature mRNA is formed 5) Export - the mRNA leaves the nucleus to be translated |
| What can happen if termination does not occur? | 1) It can interefere with transcription of a downstream gene 2) Wasting polymerase - polymerases are recycled |
| What is the principle reason why processing of mRNA is required? | Stability |
| What does the promoter act as? | Acts as a template for the assembly of a multi-component complex called the preinitiation complex, which brings pol II to the gene. Once bound the polymerase can then transcribe the gene. |
| What does UTR stand for? | Untranscribed region |
| What is the AATAAA region after the 3'UTR? | Involved in telling the machinery to make a polyA tail. |
| Describe the features of promoter architectures? | Polymerase positioning elements (TATA box, initiator, distal/upstream (and downstream) elements that bind positively and negatively acting transcription factors, called enhancers and silencers |
| How far upstream is TATAA | ~30 nucleotides upstream of the start site |
| What is a transcription factor? | Sequence-specific DNA binding proteins which can bind to regulatory elements in or near the promoter. They can bind specific sequence and interact with RNA polymerase and its supporting proteins - stabilises polymerase binding and catalyses activity. |
| What is the rate-limiting step in transcription? What affect do transcription facts have? | Binding of polymerase - increasing the transcription rate (enhancers) or decreasing the transcription rate (silencers) - i.e. transcription can be under both negative and positive regulation. |
| What is the first step of binding polII? | Transcription factor (TF2D) which includes a protein called TBP (TATAA binding protein) binds to the TATAA box. Other factors then coming in, eventually leading to the recruitment of PolII. |
| What is the transcription complex which includes polII called? What problems can it lead to? | 1) Pre-initiation complex 2) Huge (size of ribosome), difficult to access tightly wound DNA. |
| What is TF2H? | Transcription factor responsible for phosphorylating the C terminus of PolII (once bound to DNA/pre-initiation complex). This allows RNA PolII to start transcription in process called promoter clearance. |
| How do transcription factors interact with the DNA? | Either directly with members of the pre-initiation complex or indirectly via proteins called coactivators/co-factors. Either way they stabilise the formation of the pre-initiation complex and hence increase the rate of RNA PolII. |
| What determines the rate of transcription of a gene? | Rate of assembly of the pre-initiation complex. |
| What are basal factors? | Transcription factors which in response to coactivation, position RNA polymerase at the start of a protein-coding sequence and release the polymerase to transcribe the mRNA. Transcription factors interact with the basal machinery. |
| What determines tissue-specific gene expression? | Tissue specific transcription factors |
| What step occurs to move from initiation to elongation? | C terminal of RNA polII is phosphorylated by a factor that TF2A can interact with called CDK7. |
| What needs to happen for RNA polII to transcribe the DNA? How does this happen? | 1) DNA needs to unwind 2) RNA PolII has helicase activity. It is also associate with histone methylators which can modify the chromatin (proteins are bound to DNA) and are responsible for producing the transcription bubble. |
| What are the key differences in DNA and RNA? | 1) DNA is deoxyribonucleic acid - deoxyribose has lost an oxygen molecule from the 2' position from the ribose sugar (hydroxyl group in RNA). 2) Consequently DNA is much more stable (the hydroxyl group means the RNA is subject to nucleophilic attack) 3) RNA uses uracil (carbonyl group) rather than cytosine (amine group; NH2) |
| Why isn't uracil used in DNA? | Less energetically expensive than cytosine (much more RNA is produced). It is thought that RNA is the original store of genetic information (both stored and could act catalytically). The problem with using uracil as a nucleotide is that its very easy to convert cytosine to uracil - can't repair this if you are already using uracil in the genome. Therefore if cytosine is converted into uracil this can be recognised and an enzyme called uracil DNA glycosylase can repair this back to cytosine. |
| What type of structure does RNA form? | Doesn't form double helix - forms local secondary structure where there are base pairings. |
| In which direction do RNA polymerases work? | 5' to 3' |
| Describe synthesis from 5' to 3' | AT THE FIRST NUCLEOTIDE: At the 5' end is triphosphate group, at the 3' end is a hydroxyl group. The hydroxyl group forms a nucleophilic attack on the first phosphate atom (a phosphate) of the triphosphate group. This forms a phosphodiester bond, the two remaining phosphate groups (y and B) (known as pyrophosphate) are released. |
| What are the consequences of the release of pyrophosphate? | 1) It is hydrolysed which provides the energy for the reaction 2) This also means that the reaction is essentially one way. |
| How does capping work: 1) RNA polymerase 2) What capping enzymes associated with? 3) What is used to cap the mRNA? 4) What links the cap? | After the first 30 nucleotides the RNA polymerase pauses - this is because the RNA polymerase needs to process the 5' end. These 30 nucleotides then exit from the 'exit channel' of the polymerase. This means that the nucleotide strand is then accessible to capping enzymes which are associated with C terminal domain of RNA polymerase. The RNA is capped by the addition of the 5' end of the mRNA. 7-methyl guanosine is linked to the mRNA by a 5'-5' triphosphate bridge. |
| Why does the mRNA need to be capped? | The 5' end is subject to attack by exonucleases. |
| What are exonucleases? | RNAase molecules which can attack the mRNA from the 5' end. |
| What happens if the capping doesn't take place? In what way is capping specific? | 1) The RNA polymerase will not continue transcribing 2) Capping only occurs with RNA PolII transcribed molecules |
| What are the capping enzymes? | One has two activities - 1) Before the addition of the 7-methyl guanosine it removes the final phosphate group (5' phosphate activity) - creates the correct linkage format 2) The second activity is as a guanyl transferase - adds the 7-methyl guanosine (capping) 3) The other activity required is methyl transferase - creates methyl group on the prime carbon of the second ring of 7-methyl guanosine and then in the nucleus it methylates the oxygen attach to the 3 carbon of the first base, and in the cytoplasm it does the same thing to the second base. |
| X | |
| What is methylation particularly important for (and capping etc) | Stability and also translation |
| What is the rate of transcription? | 30 nucleotides per second |
| What effect on downstream genes does a failure to terminate have? | Affects their transcriptional regulation |
| How does termination occur? | 1) At the 3' end is the stop codon (AAUAAA). 2) This recognised by CPSF (a protein associated with the C terminal domain of the RNA PolII. 3) Between the AAUAAA and a uracil-rich region of the RNA is the polyA site. This is the site which is cleaved. 4) CsF (Cleavage stimulating factor) which is also associated with the C terminal recognises the uracil-rich region. 5) The CPSF and CsF recognise their respective regions and bring in cleavage enzymes which cut the RNA at the PolyA tail and recruit PAP (polyA polymerase). |
| What does CPSF? | Cleavage and polyadenylation specificity factor. |
| What is the function of PAP? What stimulates the activity of PAP? | 1) PolyA polymerase adds lots of A to the RNA molecule where is has just been cut to create the polyA tail. 2) PABII - regulates activity of PAP and length of PolyA tail |
| What is the length of the polyA tail usually? | ~200 nucleotides long |
| What is the function of the polyA tail? (2) | 1) Stabilise the mRNA 2) Aids in translation |
| How is transcription actually terminated? | Not clear why RNA PolII stops, two theories (not mutually exclusive): 1) Possible change in proteins associated with the RNA polymerase when it goes beyond the cleaved region (i.e. from enhancers to inhibitors of elongation) 2) The accessible 5' end of the remaining RNA strand is going to be cleaved/degraded by endonucleases, eventually reaching the RNA molecule and knocking it off the DNA. The C terminal is then dephosphorylated and the RNA PolII is put back into circulation and can be used for another round of transcription. |
| How many nucleotides can RNA polII go on for if not terminated? | ~1000 nucleotides. |
| What is heteronuclear RNA? | 5' cap, 3' polynucleotide tail but needs splicing. |
| How long is an exon typically? An intron? How many introns are there per gene? | 1) 150 nucleotides 2) 3 kilobases 3) 8-9 per gene |
| What process is splicing really? | A code transcriptional process because the initial splicing machinery is associated with the RNA polymerase. |
| What does the splicing machinery recognise? How common is the sequence at the start of an intron? | Splice sites - at the boundary of an exon and an intron. The final two nucleotides at the exon 3' end are A and G and the first 5' end nucleotides of introns are G and U (95%). |
| What does splicing tend to occur in? What is the molecular machinery called and what is it made up off? | 1) Splicing tends to occur in pairs - usually the first splice site is the first ones to be recognised. 2) snRNPs - small nuclear ribonuclear proteins bind splice sites. Contain both protein and RNA. |
| How is an intron removed? (7) | number of snRNPs bind to splice sites: 1) U1 snRNP binds to the 5' site 2) U2 snRNP binds to the branch point in the intron (an A nucleotide) 3) U1 attracts U4, U5 and U6 (which are in a complex) 4) U4, which is stopping the catalytic activity of U6, then leaves with U1 5) U6 can then form a bridge with U2 (lariat formation) 6) The 5' splice site can then attack the 3' splice site, resulting in the loss of the intron which is degraded 7) The two exons are ligated |
| How many genes are there? | 20,000 |
| How big is the human proteome? | 100,000 |
| Why is splicing so important? | Alternative splicing gives rise to genetic variation |
| What % of genes are thought to be alternatively spliced? | 95% (and this number is thought to be increasing) |
| What determines alternative splicing? | Splicing enhancers and suppressors which are tissue specific - can control which introns are removed and which exons are ligated together. |
| What is tropomycin involved in? What does its alternative splicing lead to? | Actin myofilament formation (primary controller of cell morphology) - can be spliced to produce actin in striated muscle, smooth muscle, various proteins in the brain and fibroblasts. |
| What happens after alternative splicing? | Mature mRNA needs to leave the nucleus. |
| How does the mature mRNA leave the nucleus? Which molecules recognise it? | The polyA tail, the 5' cap and the RNA itself can all be recognised by a number of different factors such as NxT1 and Nxf1 will bind to the RNA, along with CBC (cap-binding complex). Other proteins will bind to the polyA tail which then loop the protein round by interacting with CBC which allows the RNA molecules to be transported out of the nucleus into the cytoplasm through nuclear pores. |
| What is the relationship between transcriptional activity and nuclear pores? Roughly how many nuclear pores would you expect to find in a highly transcriptionally active cell? | 1) The more activity the more nuclear pores 2) ~4,000 |
| What are nuclear pores? | Octameric ring structures which fit through the two nuclear membranes to create a channel through which export and import as things as large as ribosomes will take place. mRNA export is regulated process. |
| What happens to mRNA once it is in the cytoplasm? | The translational machinery can recognise things which haven't been properly spliced - results in nonsense mediated decay. |
| What confers the biological activity of a cell? | The proteome |
| How do specialised cells express the appropriate enzymatic activity? | Tissue specific expression of the genome. |
| What can mutations in transcription factors and splice sites cause? | Affects gene expression and can cause disease - for example cancer (many oncogenes are mutated transcription factors, for example mutated CMIC can cause an increased rate of transcription). |
| What happens if you don't splice out an intron correctly? | This can shift the reading frame and what was normally read as an amino acid could be read as a stop codon (leads to nonsense-mediated decay, early truncation, and loss of protein). |
| What is systemic lupus erythematosus ? | Antibodies against the snRNPs (against the spliceosome) |
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