Chapter 5: Transcription pt II Flashcards
MG CAP
Carboxyl tail domain (CTD) of RNA polII serves as binding site for capping enzymes.
The MG cap is later recognized by proteins involved in the initiation of translation.
Mechanism of gene splicing
- The 5’ end of the intron sequence is GU and the 3’ end is AG.
- The splicing apparatus = proteins + snRNAs= (snRNPs).
- snRNPs + primary transcript+ proteins = spliceosome.
- Mutations in snRNA in yeast prevent splicing.
- snRNAP
1. recognizes and binds (sequence-sequence complementarity) consensus sequences in mRNA
2. Adenine (A) branch point within intron—> transesterification
Why introns? 3
- Important in gene regulation (rate of transcription)
- Evolutionary relics from gene-gene fusions
- alternative splicing (1 gene—>many products)
RNA editing
-RNA editing plays a major role in regulating the expression of genes in the mitochondria of trypanosomes and plants.
-An alteration of the information content of mRNA molecules.
-By inserting or deleting uridine bases. Discovered in mitochondria of trypanosomes.
-It is mediated by guide RNAs transcribed from other mitochondrial
RNA editing.
1) by insertion/deletiion of bases (guide RNA)
2) by changes in mRNA bases (deamination)
miRNA
- normally transcribed, bind complementary mRNA , shut down gene expression (eg. lin14 +lin4 C.elegans)
- link to diseases, e.g.. liver tumour have a specific (miRNA26) miRNA down—>deliver
- mammary tumour have miRNA10 up—>inhibit
Why do we care about proteins? 4
1) They carry metabolic processes
2) Serve as cell structural elements
3) Carriers of information within and between cells
4) DNA replication, transcription, splicing, etc need proteins.
Protein synthesis Initiation (prokaryotes)
- Binding of mRNA to 30S Subunit is stimulated by IF3 and IF1.
- IF2 binds to an initiator tRNA (fMet-tRNA) AUG is the codon for initiation of translation.
- IF2-(fMet)tRNA binds the first Codon
- A ribosomal protein catalyzes a GTP mediated reaction for the assembly of the 50S subunit.
- IFs are released
Pro. Ribosomes
Small RNAs can be used to block translation by binding to Shine-Dalgarno sequence
Protein synthesis initiation in eukaryotes
1) Ribosome small subunit binds mRNA methylated cap.
2) Ribosome small subunit moves towards AUG initiator codon
3) tRNA (anticodon) recognizes and binds AUG
4) Large subunit is recruited.
Protein synthesis: Elongation
1) EF factor is activated by GTP binding. The EF(GTP)-tRNA complex binds the A site of the ribosome.
2) GTP hydolysis allows aa-tRNA binding and EF is release.
3) Peptidyltransferase catalyses the transfer of the polypeptide chain to the aa-tRNA forming a peptide bond.
4) Other EF mediates a 5’3’ move of the peptidyl-tRNA from the P to the E site (a GTP mediated reaction), releasing the uncharged tRNA.
Protein synthesis: Termination
A stop codon is recognized by a protein release factor (RF).
•RF binds the stop codon in the A site leading to the release of the polypeptide chain from the P site and dissociation of the ribosome
Protein separation/Identification
2D gel electrophoresisallows separation of proteins in a mixture by their isoelectric point (pH at which charge is zero) and molecular weight. Spots in a 2D gel are excised and digested into peptide fragments. Fragments separated by mass spectrometry into a series of signal peaks (peptide mass fingerprint). A peptide mass fingerprint is matched with the mass spectrum of predicted proteins from a genomic DNA or translated transcriptome (mRNA)
Ribosomes
A, P, E (A-anticodon-codon; p-peptydil transferase; e-exit)
Proteomics
- prediction from genome sequence (ORF)
- 2 dimensional gel electrophoreses (2DE). Separation of proteins from a biological sample base on IP (pH at which change of protein=0…protein has no charge). MW (size)
digest protein spots from the gel into peptide fragments (trypsin)
separate fragments by mass spec—>protein fingerprint (Does it match any fingerprint from a genome predicted protein?)
