Briefly explain transport through the nuclear pore
POST TRANSCRIPTIONAL CONTROL MECHANISM
• nuclear envelope is perforated by pores that can selectively transport macromolecules across the membrane; only functional mRNAs are exported
• recognizes only 5’ cap before transported; nuclear mRNPs are exported with mRNAs
• may also be linked to splicing (prevents unspliced mRNA from being transported)
• e.g., HIV virus is only exported to cytoplasm if fully spliced
Briefly explain localization of mRNA to specific regions
POST TRANSCRIPTIONAL CONTROL MECHANISM
• use cytoskeleton to position mRNA close to sites where protein proceed by mRNA is required
• transport to specific regions involves specific RNA binding proteins
• e.g., mRNAs in learning and memory; localization of beta-actin mRNA to the leading edge of fibroblasts
Briefly explain RNA-editing; alteration of mRNA sequence
POST TRANSCRIPTIONAL CONTROL MECHANISM
n protozoans: addition or deletion of U-residues, causes changes to many nucleotides; directed by small guide RNAs (gRNA) enzyme cascade
• in mammals: site-specific single nucleotide changes; deamination of A to I (inosine), C to U
• e.g., C to U in intestine mRNA causes codon to change into stop codon, resulting in shorter Apo protein
Briefly explain gene silencing by small RNA molecules (siRNA or miRNA)
POST TRANSCRIPTIONAL CONTROL MECHANISM
• RNA interference – ability of small, double or single stranded RNA molecules to induce sequence specific gene silencing in complementary regions
• involved in regulation of expression of endogenous genes (for development/differentiation), defense against viral infection, suppression of transposon mobilization, etc.
• e.g., larval protein in C. elegans only translated in early development; mRNA still present later in development but untranslated due to lin4 small RNAs that silence genes
Briefly explain translational control switch
POST TRANSCRIPTIONAL CONTROL MECHANISM
• opposing regulation of two coordinated products with single control element that is sensitive to certain environmental conditions
• e.g., ferritin (Fe storage protein) and transferrin receptor (responsible for transport of Fe into cell) are coordinated by IRE element that binds iron-responsive binding protein; when iron is low in the cell, IRE-BP binds to elements, preventing translation of ferritin mRNA so Fe is not stored and preventing degradation of transferrin product to bring Fe into cell)
Briefly explain RNA stability (degradation and stabilization)
POST TRANSCRIPTIONAL CONTROL MECHANISM
• balance of degradation and synthesis determines the level of individual mRNAs in cells
• stabilizing elements and instability elements in 3’UTR and in coding sequence
• e.g., maternal mRNA Hsp30 is not detectable until after mid-tailbud stage
Explain the role of mRNA stability in control of gene expression
- balance between mRNA degradation and synthesis determines level of individual mRNA in cells
- presence of stabilizing elements in 3’UTR and coding sequence
- instability elements (predominantly found in 3’UTR) usually determines the rate of degradation by interacting with specific RNA binding proteins
Explain the role of RNA editing in control of gene expression
- changes to nucleotide sequence are directed by gRNA; can be large-scale or single nucleotide change
- changes will produce different product or not produce product at all
Explain the role of iRNA in control of gene expression
- RNA interference induces gene silencing
* can prevent the formation of viruses, prevention of mRNA into protein, etc.
Explain the role of transcriptional control switch in control of gene expression
• control element either prevents degradation or translation of mRNA product, controlled by environmental conditions
Explain the role of mRNA localization in control of gene expression
• localization of mRNA allows for certain protein product to be in high or low concentration in certain region
Explain the connection between pre m-RNA splicing and transport of mRNA from the nucleus.
- pre-mRNA splicing includes addition of 5’ cap; 5’ cap is recognized by transport machinery (embedded in nuclear membrane)
- special proteins that recognize processed mRNA attach (likely involved in transport membrane molecule recognition as well); get replaced by cytoplasmic mRNP as it travels through membrane
- cell has system that prevents the exit of unspliced mRNA
o proof: HIV makes only a few mRNAs that get fully spliced
o one includes REV mRNA (reverse transcriptase enzyme) that goes back into the nucleus and helps transport unspliced/unprocessed mRNA across nuclear membrane
What is trans-splicing? Give an example.
- construction of mRNA by splicing of separate RNA molecules, joining the exon of one gene with the exon of another
- e.g., in Trypanosoma:
o have leader sequences that give rise to leader exons (which get capped, but not processed at their 3’ ends)
o also have coding regions for polycistronic primary transcripts (no introns)
these get spliced into exons that code for protein; leader exons + primary transcripts = transplicing + polyadenylation of 3’ end
What does S in 16S stand for? What is the numerical value of this constant?
- S stands for Svedverg units, which is a measure of sedimentation rate (velocity) of suspended particles when centrifuged under constant conditions; velocity depends on both size and shape of the particle
- good measure of relative size (units are not additive)
- 1 S = 10-13 s
What are the roles of three major RNAs in protein synthesis?
- mRNA – carries genetic information from DNA in the form of “codons”
- tRNA – translates mRNA code; each amino acid has its own tRNA
- rRNA – associates with proteins to form ribosomes which catalyze the assembly of protein chains
How many different tRNAs are there in a eukaryotic cell? How many different aminoacyl tRNA synthetasis are there in a eukaryotic cell?
- around 50 different tRNAs in animals and plants
- there are 20 different aminoacyl tRNA synthetases (one for each of the amino acids)
What are the roles of tRNA in translation?
- to be linked to a particular amino acid (brings amino acid)
- to recognize codon in mRNA so that corresponding AA can be added
- transfers amino acid from surrounding cytoplasm to corresponding order in amino acid sequence, where anti-codon base pairs with codon; 3’ terminal site fits specifically to amino acid that corresponds with anticodon’s codon
What is the wobble position for an anticodon? For a codon?
- wobble position is non-standard pairing in tRNA
- for a codon, wobble position in the 3rd position
- for an anti-codon, wobble position in 1st position
Explain what does it mean when we say that the code is degenerate? Codons are synonymous?
- code is degenerate because there is more than one codon corresponding to an amino acid (64 codons for 20 aa’s)
- synonymous codons are codons that code for the same amino acid (typically differ in nucleotides at the third position)
How are ribosomal subunits held together? What could be used to separate subunits?
- by divalent cations (e.g., Mg)
- EDTA and high salt concentrations cause the ribosomal subunits to dissociate
