Term
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Definition
All the encoded proteins in a genome.
The proteome is not all expressed all at once |
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Term
| How is reverse transcription utilized in a lab setting? |
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Definition
| It is useful to analyze the DNA that codes protein. mRNA is in too small amounts, so by using reverse transcription there can be enough DNA to analyze |
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Term
| Describe the 2D gel technique for proteome analysis |
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Definition
1) Isoelectric focusing gel separates protein by pH
2) The gel is placed sideways on SDS for electrophoresis to seperate by molecular weight |
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Term
| What is a disadvantage to 2D proteome gel analysis? |
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Definition
| Hydrophobic proteins are insoluble in the gel |
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Term
| Two fundamental processes involve DNA and two involve RNA in bacteria and mammalian cells. What are they? |
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Definition
DNA: Replication and transcription
RNA: Transcription and translation |
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Term
| Give four important features of the structure of RNA. |
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Definition
a) Contains ribose – no 2-deoxyribose
b) Has uridine instead of thymidine
c) Single stranded
d) Alkali-labile |
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Term
| What are the 4 requirements of DNA dependent RNA polymerase? |
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Definition
a) Core enzyme
b) DNA strands,
c) Mg & Zn ions,
d) NTPs |
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Term
| To what do the terms ‘template strand’ and ‘coding strand’ refer? |
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Definition
The ‘template strand’ is a DNA strand that is transcribed to RNA.
The ‘coding strand’ is the DNA strand complementary to the ‘template strand’ and whose nucleotide sequence is identical to the RNA transcribed from the template strand |
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Term
| In what direction does RNA polymerase extend a chain? |
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Definition
| The new chain grows from 5’ to 3’ [The ribose 3'-OH group of the previously present ribonucleotide attacks the next (incoming) complementary ribonucleotide triphosphate molecule, removing pyrophosphate (PPi) and adding to the chain at the 3’ end]. |
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Term
| What is a TATA box and why is it not transcribed? |
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Definition
| A short sequence of thymidine and adenine residues that promotes gene transcription. It lies about 10 nucleotides upstream of the first transcribed nucleotide and binds to sigma factor (σ factor) in bacteria. In eukaryotes it lies slightly further upstream and binds to TATA binding protein. The two DNA strands become separated downstream of the TATA box on the coding strand by helicase action of the RNA polymerase holoenzyme. |
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Term
| The bacterial RNA polymerase core enzyme has 4 subunit polypeptides, alpha, beta, beta' and omega. What is the function of each subunit? |
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Definition
Alpha – assembly;
Beta – catalysis;
Beta’ – binding to template;
Omega – chaperone |
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Term
| What is the function of the RNA polymerase signal factor? |
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Definition
| Sends the core enzyme to the promoter, a sequence upstream of the region of DNA to be copied |
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Term
| Why is the bacterial enzyme with sigma factor referred to as a holoenzyme, not the core enzyme? |
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Definition
| Because sigma factor dissociates when the enzyme is catalyzing the synthesis of RNA |
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Term
| What is the immediate result of successful promoter function? |
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Definition
| Separation of the two DNA strands within the RNA polymerase holoenzyme |
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Term
| How does the sigma (σ) protein function? |
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Definition
| Sigma binds promotor sequences on DNA to initiate RNA synthesis |
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Term
| How does the rho (ρ) protein function? |
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Definition
| Rho binds to a Cytidine-Adenine-rich terminator sequence on the newly synthesized RNA and this binding causes the RNA-DNA complex to isomerize and dissociate, terminating RNA synthesis |
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Term
| Describe the transcription bubble |
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Definition
| A 17bp long bubble between the two DNA strands in transcription 8bp form a hybrid and 9 provide space for the new RNA to be pulled from the hybrid |
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Term
| How does RNA transcription editing differ between bacteria and eukaryotes? |
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Definition
| No proofreading in bacteria, eukaryotic polymerases will pause for a mis-paired base to fall away |
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Term
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Definition
| An enhancer region of bacteria DNA in the -40 to -60 region. Binds to the alpha subunit for strongly expressed genes |
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Term
| Describe transcription termination in E. coli |
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Definition
1. AAA and UUU sequences form a stem loop that causes isomerization and termination (or it could escape)
2. Rho protein binds to the sequence, causing isomerization, dissociation, and termination |
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Term
| In eukaryotes, what are the functions of each of the 4 core RNA polymerase enzymes? |
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Definition
RNA Pol 1: transcribes rRNA
RNA Pol 2: transcribes mRNA
RNA Pol 3: transcribes tRNA and small RNA
RNA Pol 4: transcribes mitochondrial DNA |
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Term
| Indicate three ways in which the process of transcription is more complex in eukaryotes than in prokaryotes? |
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Definition
a) Different polymerases for different types of RNAs
b) Many additional accessory protein factors
c) Core enzyme has additional C-terminal domain (CTD) that gets phosphorylated |
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Term
| Describe three characteristics of a prokaryotic mRNA transcript that differentiates it from a eukaryotic transcript? |
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Definition
| Prokaryotic mRNA transcripts have no introns, are not processed and are translated immediately because they are rapidly degraded |
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Term
| Describe actinomycin's mode of action |
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Definition
| Actinomycin D inhibits all RNA transcription because it binds to certain (GpC) sequences in DNA, preventing the open transcription complex from forming. |
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Term
| Describe rifampicin's mode of action |
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Definition
| Rifampicin binds to the β- (catalytic) subunit of bacterial RNA polymerase and is therefore an antibiotic. |
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Term
| Describe α-amanitin's mode of action |
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Definition
| α-Amanitin binds strongly to eukaryotic RNA Pol II but not bacterial RNA polymerase and is therefore a poison |
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Term
| Assuming that siRNA and miRNA are subsets of scRNA, name the other 4 RNA types in a eukaryotic cell and order them from greatest to least amounts present. |
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Definition
| rRNA > tRNA > mRNA > snRNA |
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Term
| Which RNA type is absent from prokaryotes and why? |
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Definition
| snRNA; there are no introns to be processed in bacterial mRNA |
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Term
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Definition
| Carboxy-terminal domain, the largest subunit in Pol II Has phosphroylation sites involved in transcription |
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Term
| How much of human DNA is genes? How much of bacterial DNA is genes? |
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Definition
| 1.5% in humans, 90% in bacteria |
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Term
| What is the function of mRNA? |
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Definition
| template for the amino acid sequence of a polypeptide |
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Term
| What is the function of tRNA? |
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Definition
| activates amino acids and incorporates them into a polypeptide by reading the genetic code on the mRNA |
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Term
| What is the function of rRNA? |
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Definition
| complexed with proteins in ribosomes, platforms for translation interaction of tRNA with mRNA and catalyze peptide bond formation |
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Term
| What is the function of snRNA? |
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Definition
| (Small nuclear RNA) ribonucleoprotein particle that excises introns from primary mRNA transcripts in the eukaryotic nucleus – absent from prokaryotes |
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Term
| What is the function of scRNA? |
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Definition
| Small cytoplasmic RNA helps move polypeptides for synthesis in the endoplasmic reticulum lumen |
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Term
| What is the function of RNAi? |
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Definition
| Composed of small interfering RNA (siRNA) and micro RNA (miRNA). RNAi synthetic pathways use an RNase (dicer) that generates siRNA or miRNA from long dsRNA or short hairpin RNA (pre-miRNA) |
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Term
| What is the function of siRNA? |
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Definition
| siRNA binds viral RNA and targets it for destruction; miRNA binds to and eliminates host mRNA during animal and plant development |
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Term
| What is the most prevalent type of RNA in a eukaryotic cell? |
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Definition
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Term
| Freshly synthesized eukaryotic mRNA is processed at what 3 sites? |
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Definition
| Sites are the 5’-end, the 3’-end and the middle: i.e. the cap, polyA tail and intron excision |
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Term
| How does each complex recognize the portion of mRNA that it acts on? |
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Definition
| Cap synthesizing complex recognizes the 5’-end triphosphate The endonuclease of the polyA synthesizing complex and the snRNA particles of the spliceosome complex recognize consensus sequences on the remainder of the unprocessed mRNA |
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Term
| What three enzymes are involved in 5' cap formation? |
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Definition
1. Phosphohydrolase (removes Pi from 5' end)
2. guanosyl transferase (activates GTP and adds it to end)
3. methylase (Adds methyls to the N7 atom) |
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Term
| What allows mRNA to enter the cytosol? |
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Definition
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Term
| What enzymes are involved in mRNA 3' tail formation? |
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Definition
| Endonuclease (cleaves downstream of consensus site) polyA polymerase of the poly A polymerase complex (Adds many ATP to make the tail) |
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Term
| How does mRNA splicing occur? |
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Definition
| U2 RNA attacks U1 RNA, forming a lariat with with associated proteins that the spliceosome complex then excises |
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Term
| What are the functions of exon-intron 5’ and 3’ boundaries and of a downstream adenosine residue near an intron’s 3’end? |
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Definition
| These three regions possess consensus sequences, nucleotide residues that hybridize with sequences on various snRNAs necessary to excise an intron |
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Term
| What nucleotide is the substrate for the first endonuclease action of the spliceosome complex? What are the spliceosome products? |
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Definition
| An adenylate residue within the intron being excised; Processed mRNA with the exon plus the intron excised as an RNA lariat |
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Term
| One gene can encode two different polypeptides. How? |
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Definition
| Alternative splicing, or partial proteolysis |
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Term
| What enzymes are required to process freshly synthesized tRNA and rRNA? |
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Definition
| Specific endo- and exo-nucleases that trim the ends and may also remove an intron without requiring the snRNA spliceosome complex |
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Term
| How long is the turnover of mRNA in prokaryotes? How long in eukaryotes? |
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Definition
1.5 minutes in prokaryotes
Variable in eukaryotes (average 3 hours) |
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Term
| How is RNA degraded in eukaryotes? |
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Definition
| Specific endo- and exo-nucleases that trim the ends and may also remove an intron without requiring the snRNA spliceosome complex |
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Term
| What are the functions of the cap and tail in eukaryotic mRNAs? |
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Definition
| The cap facilitates splicing. The tail facilitates mRNA transport out of the nucleus. Both facilitate the initiation of translation by attaching to the ribosomal small subunit. Cap and tail are also the sites of initial mRNA degradation. |
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Term
| How might you determine whether a given gene is likely to be expressed by a eukaryotic cell at any given time and/or in a given environment |
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Definition
| All synthesized proteins have mRNA with a poly-A tail which hybridizes to a poly-U or poly-dT column. The bound mRNA can be eluted with high salt, southern blotted, and probed with a radioactive nucleotide sequence that encodes part of the protein of interest. If the probe binds to the mRNA blot, the presence of the appropriate mRNA will beindicated by a radioactive spot |
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