Measured by density gradient centrifugation. S value of a molecule determined by mass and structure of a molecule.
Roles of rRNA
Structural element Translation initiation Catalyzed the peptide bond formation
How do antibiotics work?
Many work on by specifically binding and inhibiting prokaryotic ribosomes
1. Carring the amino acid at its acceptor arm 2. decoding the codon 3. Help the peptide bond formation
tRNA structure Acceptor Arm, D Loop L Loop Variable Loop Anticodon-loop
Arm - 5' CCA 3' holds amino acid D-loop contains two dihydrouridine T-Loop - contains pseudouridine Variable loop - varies 4-13nt Anticodonloop - bases of anticodon flip out and often contain inosine (for wobble pairing)
What prevents different open reading frames from happening?
Frame shifts often have many stop codons that prevent copying of frame shifts
64 tubes containing one codon. With other translational material and one of 20 amino acids. Pass each reaction on a NC filter. Only ribosome-mRNA-tRNA amino acid complex can bind to NC filter. If the radioactive maino acid is recognized by and attached to the correct tRNA, it was radioacitvely labeled. 20 sets of 64 reactions required to break genetic code.
Stop codons and initiation codons
Stop: UAA UGA UAG Initiation: AUG
Define degeneration Which amino acids are degenerated? Which are not?
Genetic codes are redundant. Arginine, Leucine, Serine are all degenerated the most Methioinine and Tryptophan are not degenerated (Methionine is the first one)
Wobble base pairing and how is this accomplished?
One anticodon of tRNA can recognize and base pair with more than one degenerate codons encoding the same amino acid Mismatching U with A or G or G with U or C Or Inosine, can base pair with A, C and U
Aminoacyl-tRNA, how is it made? and where does amino acid attach to tRNA
Aminoacyl-tRNA synthetases (ARS) attaches amino acid to tRNA. Cells have 20 ARS recognizes tRNA by its acceptor arm. ARS proofreads and remove mistakenly incorporated residues Using ATP, ARS covalently attaches amino acid to the 5' phosphate group of AMP ARS transfers amino acid from AMP to 3' -OH group of tRNA
Prokaryotic translation initiation
Starts after the Shine Dalgarno Sequence (SDS) 5bp upstream from translation initiation AUG. Base pairs with 16S-rRNA. Can also use S1 to determine initiation Can have multiple SDS, all work independently Initiation factors (1) prevent two free ribosome subunits from binding to each other and (2) help assemble 30S initiation complex. Always starts with formylated methionine
Eukaryotic translation initiation
First Kozak sequence always used, AUG part of Kozack. First met is often acetylated. Initiation factors preventing bonding of subunits and also help initiate translation.
Eukaryotic initiation factor 4 eIF4E - binds to cap eIF4G - adaptor protein can bind to many other proteins, and binds to polyA tail, creating circular mRNA eIF4A - RNA helicase activity eIF4H - stimulates eIF4A helicase cativity
Regulation of translation initiation mTOR, insulin
5' UTR plays an important role. Secondary structure at 5' UTR may bind to a regulator protein. also regulated by initiation factors When insulin is present, it activates a kinase called mTOR. mTOR phosphorylates PHAS-1 which does not bind to eIF4E. Free eIF4E binds to cap of mRNA and activates initiation
CPE Maskin, controlling translation
CPE of cyclin B binds to CPEB. CPEB brings in Maskin and masking binds to eIF4E, replacing eIF4G and turning off trnaslation. When the oocyte is activated, a kinase called Eg2 phosphorylates CPEB which brings in CPSF (Poly adenylation specificity factor) and changes conformation of Maskin, releasing eIF4E. eIF4E binds to eIF4g to turn on translation.
APE, in that order The first amino acid arrives in P site, The first amino acid to attach to that enters A Site. Peptide formation catalyzed by peptidyl transferase. Believed that 23S or 28S rRNA has catalytic properties. New aminoacyl enters A site. Peptide bond is formed between A site and P site. The P site is now empty, and ribosome moves toward the 3' end of mRNA, and each tRNA moves over one.
When the stop codon hits, no tRNA enters the A site, so RF/eRF1 have a structure similar to tRNA, so they can fit into the empty A site. RF1/eRF1 promotes cleavage of the last peptidyl tRNA on the P site, and all the components fall apart and recycle
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