Name parts of the machinery that drives translation: specifically: ribosomes, mRNA, tRNA, aminoacyl tRNA synthetases, initiation factors, elongation factors, and release factors.
(7) ribosomes, mRNA, tRNA, aminoacyl tRNA synthetases, initiation factors, elongation factors, and release factors.
What is the role of ribosomes, how many subunits/binding sites do they have?
The ribosome contains the catalytic center, contains both RNA and many proteins.it contains two subunits: in bacteria these are the 30S and 50S, and in eukaryotic these are the 40S and 60S. In a fully assembled ribosome, the mRNA and tRNA pass between the two subunits;
there are three tRNA binding sites: the A, P, and E sites.
What does the small (40S) ribosome subunit do?
The small subunit has the decoding groove through which the mRNA passes and the tRNAs read the message.
What does the large (60S) ribosome subunit do?
The large subunit contains the catalytic center (the peptidyl transferase center, PTC), which appears to be made entirely of RNA and thus the ribosome is a ribozyme (uses RNA to perform catalysis).
What is the role if mRNA
contains the nucleotide sequence that encodes the protein. There are 64 (4^3) possible codons, all are used.
What is the role of tRNA (and what are the main parts of the tRNA that are involved in performing that role)
adapters that “read” the message and deliver the right amino acid. tRNAs base-pair directly to the codons in the mRNA though the the tRNA’s anticodon loop. the acceptor stem has the amino acid attached that matches the anticodon. Some tRNAs can recognize more than one codon, due to wobble-pairing at the third location in the codon. The amount of each type of tRNA in the cell varies, usually matching codon frequency. The function of tRNA is dictated by its three-dimensional folded structure.
What is the role of aminoacyl tRNA synthetases
protein enzymes that put the right amino acid on the right tRNA.
What is the role of initiation factors
They bring the ribosome to the message and assist in getting the machinery assembled.
What is the role of elongation factors
They deliver tRNAs and move the ribosome down the message.
What is role of termination factors
They end the process at a stop codon, and dissociate the subunits so they can be used again..
What does degeneracy mean with regards to the genetic code?
there are multiple codons for a single amino acid
Describe Initiation in bacteria
the ribosome binds essentially right at the start codon due to the Shine-Dalgarno sequence and three initiation factors work to assembly the full ribosome.
Describe initiation in eukaryotes
initiation factor (eIF) 4E is required to bind to the 7-methyl guanosine cap on the 5’ end of the mRNA. This leads to binding of many other eIFs (4G, 4A, 4B, etc.) and eventually to binding of the small ribosomal subunit, which itself is bound by several factors (eIF3, eIF1A, eIF1, eIF2, etc.). The ribosome then scans down the message to find the AUG start codon. At that point, the large subunit can join the small, the factors are released, and the goal of initiation has been achieved.
Describe the elongation process (cycle)
aa-tRNA enters A-site (delivered by EF1A in eukaryotes, EF-Tu in bacteria), where its anticodon loop base-pairs with the right codon in the mRNA, “reading” the message. The peptide bond forms when the protein chain moves form the P-site tRNA onto the A-site tRNA. elongation factor catalyzes translocation, in which the message and tRNA move one codon over. Now, the empty tRNA form the P site is in the E site and the nascent peptide chain is attached to the tRNA in the P-site. This leaves the A site open and the cycle repeats for each codon.
Ribosome reads a stop codon at the end of the sequence, ending elongation, and dissociating the subunits. A stop codon is detected by a recycling factor in the A-site. Recycling factors are proteins that fit into the same space as a tRNA, but when they do, they trigger the termination of the peptide chain, and a series of events that lead to release of the peptide and dissociation of the subunits. The protein then goes off to fold, receive any modifications, etc.
Describe the recycling step
ribosomal subunits are prepared to be used again in initiation
What is cap independent initiation and what is the significance
a cap-independent process in eukaryotes is driven by specific RNA sequences and structures called internal ribosome entry sites IRES. IRESs are used by viruses, but it also appears that this process can be modulated based on the cellular conditions and thus they are probably very important for regulating gene expression.
what is interferon stimulation and why is it significant
interferons are generally released when a cell in infected with a virus. interferons can phosphorylate eIF2-alpha. when this is phosphorylated,it’s activity, binding of the initial tRNA is blocked and translation so blocked. This could be a self protection mechanism of the cell during infection
What is mRNA editing and why is it significant
the transcribed mRNA is modified specifically in such a way that the coding is affected. This can be tissue specific, so the same gene encoded in DNA can be use to produce two different proteins. apoB is a good examples.
What is rapamycin treatment and why is it significant?
Rapamycin causes a dephosphorylation of 4E BP (binding proteins) allowing them to bind to eIF4E (the cap binding protein) inhibiting cap dependent translation initiation. (side note: mTOR induces phosphorylation of the 4E - BPs)
Why is eIF2-alpha phosphorylation important?
eIF2-alpha is an initiation factor that binds to the small subunit of ribosomes. When it is phosphorylated, its activity is inhibited. This phosphorylation can be triggered by interferons
What are some antibiotics that function by inhibiting translation?
steoptomycin, Erythromycin, Tetracycline, Chloramphenicol (affect ribosomes), Rapamycin
how do intracellular levels of iron can be regulated by translation? this is an example of how protein-mRNA interactions regulate translation.
The iron response element is a loop structure in certain mRNA (like transferrin receptor that helps bring iron into a cell). the IRE binds to iron response binding proteins. Under high Fe conditions, IRBP binds Fe, and not mRNA, in low Fe conditions, IRBP not bound to Fe binds to mRNA at the IRE.
in case of transferrin receptor, binding of IRBP stabilizes mRNA by preventing degredation (its in the 3’ UTR). Ferritin binds to excess iron in cell, so when IRBP binds to it, in low iron conditions, it inhibits translation (the IRE is located just upstream of the start codon)