Levine Flashcards
Two fundamental processes involve DNA and two involve RNA in bacteria and mammalian cells. What are they?
DNA: Replication and transcription RNA: Transcription and translation
What four features differentiate the structure of RNA from that of DNA.
- Contains ribose instead of 2-deoxyribose
- Has uridine instead of thymidine
- Single stranded
- Alkali-labile
What are the 4 requirements of DNA dependent RNA polymerase?
- Core enzyme
- DNA strands
- Mg & Zn ions
- NTPs
To what do the terms ‘template strand’ and ‘coding strand’ refer?
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
In what direction does RNA polymerase extend a chain? What metal ion is essential for catalysis.
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]. Catalysis requires Mg+2 ions
What is a TATA box and why is it not transcribed?
The TATA box 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
The bacterial RNA polymerase core enzyme has 4 subunit polypeptides, alpha, beta, beta’ and omega. What is the function of each subunit?
Alpha – assembly; Beta – catalysis; Beta’ – binding to template; Omega – chaperone
What is the function of the sigma cofactor in RNA polymerase?
Sends the core enzyme to the promoter, a sequence upstream of the region of DNA to be copied
Why is the bacterial enzyme with sigma factor referred to as a holoenzyme, not the core enzyme?
Because sigma factor dissociates when the enzyme is catalyzing the synthesis of RNA
What is the immediate result of successful promoter function?
Separation of the two DNA strands within the RNA polymerase holoenzyme
How do the sigma (σ) and rho (ρ) proteins function?
Sigma binds promotor sequences on DNA to initiate RNA synthesis. The Rho-dependent terminator occurs downstream of translational stop codons. It is comprised of a Rho utilization site (rut), an unstructured cytosine-rich sequence on the mRNA and a downstream transcription stop point (tsp). The rut is an mRNA loading site for Rho from which it translocates down the mRNA to the stalled RNA polymerase. Contact between Rho and the RNA -DNA complex dissociates the transcriptional complex through a mechanism that involves Rho’s helicase activity
Indicate three ways in which the process of transcription is more complex in eukaryotes than in prokaryotes?
- Different polymerases for different types of RNAs
- Many additional accessory protein factors
- Core enzyme has additional C-terminal domain (CTD) that gets phosphorylated
Describe 4 characteristics of a prokaryotic mRNA transcript that differentiates it from a eukaryotic transcript?
- no introns
- not processed
- translated immediately
- rapidly degraded
Actinomycin D inhibits transcription in all organisms. Compare its mode of action with that of α-amanitin.
Actinomycin D inhibits all RNA transcription because it binds to GpC sequences in DNA, preventing the open transcription complex from forming. α-Amanitin binds strongly to eukaryotic RNA Pol II but not bacterial RNA polymerase
Define a gene, an exon and an intron.
A gene is a section of DNA that encodes a protein or an RNA molecule that serves a function other than encoding a protein. An exon is a regions of DNA that is expressed (translated) into a protein. An intron is an intervening region of DNA that is transcribed into RNA but then excised before the gene is translated
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.
rRNA > tRNA > mRNA > snRNA
scRNA would be after snRNA
Which of these RNAs is absent from prokaryotes and why?
rRNA, tRNA, mRNA, snRNA
snRNA; there are no introns to be processed in bacterial mRNA
Freshly synthesized eukaryotic mRNA is processed at 3 sites. What are the sites, and resulting alterations to the mRNA?
Sites are the 5’-end, the 3’-end and the middle: i.e. the cap, polyA tail and intron excision
Freshly synthesized eukaryotic mRNA is processed at 3 sites. How does each complex recognize the portion of mRNA that it acts on?
Cap synthesizing complex recognizes the 5’-end triphosphate (Slide 39); The endonuclease of the polyA synthesizing complex and the snRNA particles of the spliceosome complex recognize consensus sequences on unprocessed mRNA
One site of mRNA is processed using a complex of three classes of enzymes, one site a complex of two classes of enzymes and one a complex containing a single enzyme class. Name the enzyme components that act at each site.
- Phosphohydrolase, guanosyl transferase and methylase on the cap synthesizing complex
- Endonuclease and polyA polymerase of the poly A polymerase complex
- Endonuclease formed by snRNA particles of the spliceosome complex
One site of mRNA is processed using a complex of three classes of enzymes, one site a complex of two classes of enzymes and one a complex containing a single enzyme class. To what do the spliceosomes attach before acting?
A Cap Binding Complex that replaces the Cap Synthesizing Complex on the phosphorylated C-terminal domain of RNA pol II
What are the functions of the intron 5’ and 3’ boundaries and of a downstream adenosine residue near an intron’s 3’end?
The first two regions hybridize with complementary sequences on snRNAs necessary to excise an intron and the third is cleaved when the upstream exon (3’ end) is connected with the downstream (5’ end)
hat nucleotide is the substrate for the first endonuclease action of the spliceosome complex? What are the spliceosome products?
An adenylate residue within the intron being excised. Processed mRNA with the exon plus the intron excised as an RNA lariat
One gene can encode two different polypeptides. How?
Alternative splicing or partial proteolysis
What enzymes are required to process freshly synthesized tRNA?
Specific endo- and exo-nucleases that trim the ends and may also remove an intron without requiring the snRNA spliceosome complex
What are the functions of the cap and tail in eukaryotic mRNAs?
The cap facilitates splicing and the tail facilitates mRNA transport to the cytosol. Both stabilize mRNA by protecting it from degradation and are also the sites of initial mRNA degradation. Cap and tail also facilitate the initiation of translation by attaching to the ribosomal small subunit
From your knowledge of the structure of eukaryotic mRNA in slide 37, how might you determine which genes are expressed by a eukaryotic cell at any given time and/or in a given environment?
All synthesized proteins have mRNA with a poly-A tail which hybridizes (binds) to a poly-dT primer. Use reverse transcriptase with a poly-T primer and an attached red or green fluorescent dye to make cDNA from the mRNA expressed at each stage of development or in the different environments. Add each PCR product to a microarray of genomic DNA from the organism on a glass slide and let them hybridize. Red indicates genes that made mRNA at stage 1, green indicates genes that made mRNA at stage 2 and yellow (mixture of red & green) indicates mRNA present equally at both stages
