What are the three steps of protein synthesis?
Initiation, elongation, termination
How many factors are involved in eukaryotic initiation protien synth?
In eukaryotes initiation is more complex, and requires at least 12 proteins, designated eIFs (eukaryotic initiation factors).
Describe the process of baterial iniation of protein translation?
At this point the ribosome is assembled, the initial N-formylmethionyl tRNA is in place and elongation can begin.
How are the pre initation complexes formed?
There are two tRNAs for methionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence; both are aminoacylated by the same methionyl-tRNA synthase.
mRNA cap-binding protein complex eIF-4F is composed of eIFs 4E, 4G, 4A, and 4B.
4E – binds the 5’ cap
4G – binds 4E at the 5’ cap and PABP at the 3’ poly-A tail, linking ends
4B –melts secondary structure at 5’ end (ATP dependent)
How is the 48s initation complex formed?
mRNA binds to 43S preinitation complex
What is the initiation codon?
What does elF-3 do?
5’ terminals of most mRNA molecules in eukaryotic cells are capped (methyl-guanosyl triphosphate cap). This cap facilitates binding of mRNA to preinitiation complex.
eIF-3 is key –binds to 4G and links mRNA complex to 40S ribosomal subunit
The AUG initiation codon is usually the 5’ most AUG, but precise codon is determined by Kozak consensus sequences (purine at positions -3 and +4 relative to the AUG).
How is the 80S ribosome formed?
Hydrolysis of nucleotides helps to make a given step a “one-way” step – as energy is released during that step and this makes it energetically unlikely to be reversed.
What are the three tRNA binding sites of ribosmes for elongation?
The mechanism of elongation in prokaryotic and eukaryotic cells is similar.
What is the inital elongation factor and what occurs to it to allow the tRNA to bind to the A site?
The selection of the correct aminoacyl tRNA for incorporation into the growing polypeptide chain is a critical step that determines the accuracy of proteins synthesis (error rate of less than 10-3). The small ribosomal subunit has proofreading ability. The elongation factor eEF-1α is equivalent to EF-Tu in prokaryotes.
How does Diptheria toxin affect elongation?
Inactivates eEf-2, preventing elongation
How does Ricin affect elongation of translation?
Cleaves adenine residue from the 60s subunit, eEF-2 can't bind
What is the ribosome tranlocation mediated by in elongation of protein translation?
Translocation of the ribosome on the mRNA – moves one codon (three nucleotides)
What elongation factor is used to "recharge" eEF-1alpha and what does it do?
----eEF-1betay and it promotes the exchange of GDB to GTP
eEF-1βγ is equivalent to EF-Ts in prokaryotes.
This is a common step for regulating global protein synthesis.
What are the stop codons?
UAA, UAG, UAG
What terminates protein synthesis and which one recognizes stop codons?
-----Release factors and it is eRF-1
In prokaryotes RF1 recognizes UAA or UAG, RF2 recognizes UAA or UGA.
In eukaryotes eRF1 recognizes all three stop codons
How are siRNA's and miRNA's active in protein regulation?
Small interfering RNAs (siRNAs) are produced from double-stranded RNAs by the nuclease Dicer. Pair with RNA-induced silencing complex (RISC) and induce cleavage of target mRNA.
MicroRNAs (miRNAs) are transcribed by RNA polymerase II, then cleaved by nucleases Drosha and Dicer. Pair with RISC to repress translation of target mRNA and stimulate degradation by stimulating deadenylation.
What is an example of protein regulation by the capping of repressor on 5' UTR?
How does regulation of proteins work through repressing 3' UTR?
RNA is flexible.
3’ repressor binds eIF4E, inhibiting interaction with eIF4G, blocking initiation of translation.
In what cell types does localization of mRNA's have an important part of temporal and spatial translational regulation?
Localization of mRNAs is an important part of spatial and temporal translational regulation in a variety of cell types, including eggs, embryos, nerve cells, and moving fibroblasts.
What factor is responsible for transferring a new GTP to reactivate elF-2 through phosphorylation?
What blocks the exchange of bound GDP for GTP when regulating initation factors?
----phosporylation of eIF-2 and eIF-2B by regulatory kinases
eIF-2 can be phosphorylated on serine51. Modulating initiation factor activity results in global effects on overall translational activity rather than effects on translation of specific mRNAs. There are protein kinases activated when cell is under stress and the energy expenditure required for protein synthesis would be deleterious (e.g. amino acid/glucose starvation, virus infection, large quantities of misfolded proteins, serum deprivation, hyperosmolality, heat shock)
What is phosporylated to allow formation of eIF-4F?
4E also binds to 4EBP1, which prevents 4E from binding to 4G, inhibiting translation initiation. Insulin and other growth factors phosphorylate BP1, causing it to dissociate from 4E.
What role does ATP and GTP hydrolysis play in initation and elongation of protein elongation?
You will find many such examples where key biochemical steps are energetically encouraged or made essentially irreversible through hydrolysis of nucleotides.
What is the anitbiotic that affects initiation of protein translation?
Because of the differences in ribosomal structure in eukaryotes and prokaryotes, antibiotics that specifically target prokaryotic protein synthesis can be especially effective. Such antibiotics result in growth arrest or death of the prokaryote, but do not interact with components of eukaryotic ribosomes and so are not toxic to eukaryotes.
How does Pruomycin inhibit protein synth?