What are the differences between RNA and DNA?
DNA
- made up of deoribose sugars
- double stranded so its more stable
RNA
- replaces thymine with uracil
- has ribose sugar
What can RNA do?
RNA can be an intermediate between DNA and protein, a modifier of gene expression, and enzymes that often work with proteins
What is some evidence that we see in DNA processes today that support the “RNA world” hypothesis?
How RNA is needed for all complex processes, as well as how RNA increases in complexity (ribonucleoproteins) until we get to DNA.
What are some of the main differences between transcription/translation process in eukaryotes vs. bacteria?
In bacteria, translation can begin on the 5’ region of the mRNA before transcription is finished. One gene can have multiple RNA polymerases to generate many RNAs in bacteria as well. Eukaryotic RNA is transcribed inside the nucleus and sent to the cytoplasm
What is a consensus sequence?
Areas recognized by the RNA polymerase holoenzyme that come 10 and 35 bp before the promoter region.
Bacterial promoters have 2, and RNA polymerase can recognize multiple consensus sequences.
What is the significance of a promoter?
Promoters are regions of DNA that lie upstream of a gene that is recognized and bound to by RNA polymerase. They direct the polymerase to where it needs to land
What is the role of the sigma subunit?
It changes the conformation and DNA-binding specificity in RNA polymerase so it can recognize multiple consensus sequences
What is the name of the RNA polymerase structure when the sigma subunit is bound to it?
RNA polymerase holoenzyme
How is transcription commonly terminated in bacteria?
Intrinsic and rho-dependent. Both involve forming hairpin structures between complementary nucleotides in the RNA molecule and a tail of As and Ts after the hairpin
What are the responsibilities of the different RNA polymerases in eukaryotes?
- Polymerase I is for ribosomal RNA genes
- Pol II is for protein coding genes and most small nuclear RNA genes
- Pol III is for tRNA genes, one small nuclear RNA gene and one ribosomal RNA gene
Describe the two experiments used to detect where a promoter region is located.
You can introduce mutations in upstream sequences and examing the effect on transcription, or check whether a protein binds to upstream regions using DNA electrophoresis (DNA+protein is slower)
Why do shared regions across diverged species provide evidence of conservation? What does this tell us about the conserved region?
Similar regions in many different species provides evidence for conservation because it means that there is significant sequence similarity. This tells us that the region has extreme functional importance.
What type of natural selection typically drives conserved regions of DNA?
purifying selection
What are the three main post-transcriptional modifications that eukaryotic pre-mRNA experience in preparation for travelling out of the nucleus?
- 5’ capping -> after the first 20-30 nucleotides are sequenced of mRNA, a methylated nucleotide (guanine) is added to the 5’ end to prevent degradation, facilitate transfer to the cytoplasm, the splicing of introns, and increase translation efficiency
- 3’ polyadenylation -> a section of the 3’ end is replaced with a string (poly) of adenines, associated with the termination of transcription
- Intron splicing -> gets rid of the bits that are not translated
What is splicing? What are the major steps involved in splicing?
Splicing gets rid of introns (the parts of mRNA that are not translated).
1. snRNP U1 binds 5’ spice site and U2 binds to the branch site
2. snRNPs U4, U5, and U6 bind to the complex and form the inactive spliceosome. The lariat intron structure forms
3. U4 dissasociates to form the active spliceosome followed by 5’ cleavage and formation of a 2’-5’ phosphodiester bond to stabilize the lariat intron
4. the 3’ end of the intron is cleaved
5. Cleavage frees the lariat intron and the exons are ligated (together)
What three sites are recognized by the spliceosome?
The 5’ splice site, the 3’ splice site, and the branch site (becomes the lariat)
What is the lariat structure?
A structure formed during intron splicing by covalent bonding of the 5’ guanine of an intron to the branch point adenine of the intron
How is it possible that humans have such high complexity and protein variation, but very few genes?
Alternative RNA transcription and splicing, using alternative promoters, alternative polyadenylation, and alternative pre-mRNA splicing all lead to different proteins from the same DNA sequence
Why do eukaryotes have introns, whereas most bacteria do not?
Because selection is less efficient in lineages with small effective population size (Nₑ). Many introns are slightly deleterious (extra DNA to replicate/transcribe; risk of splicing errors). In multicellular eukaryotes (typically smaller Nₑ), genetic drift overwhelms weak purifying selection, so introns accumulate; in bacteria (typically very large Nₑ), purifying selection efficiently removes such small costs (“genome streamlining”). Bacteria usually have much larger Nₑ → drift is weak, selection is strong. Multicellular eukaryotes usually have smaller Nₑ → drift is strong, selection is weak.
What are the 4 steps of transcription in bacteria?
- Promoter recognition
- transcription initiation
- chain elongation
- chain termination
