Translation I and II Leaning goals Flashcards
- Name and describe the roles of the parts of the machinery that drives translation, specifically: ribosomes, mRNA, tRNA, aminoacyl tRNA synthetases, initiation factors, elongation factors, and release factors
1.ThemessengerRNA(mRNA)
Contains the nucleotide sequence that encodesthe protein. The protein that is encoded in each mRNA is written in three-nucleotide “codons.” The codons do not overlap. There are 43=64 possible codons, all are used. AUG is used as the “start” codon, and there are several used as “stop” codons. Several different codons can encode the same amino acid, but the frequency by which codons are used is not random and can vary between organisms.
2.Transfer RNAs (tRNA)
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. Thus, each codon is recognized by a specific type of tRNA. At the other end of the tRNA in the acceptor stem that 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.
Aminoacyl tRNA synthetases
Protein enzymes that put the right amino acid on the right tRNA. These enzymes are very important in that each identifies the right tRNA and puts on the correct amino acid. Each amino acid/tRNA has its own synthetase associated with it (e.g. valyl-tRNA synthetase puts a valine on a val-tRNA).
- Name and describe the roles of the parts of the machinery that drives translation, specifically: ribosomes, mRNA, tRNA, aminoacyl tRNA synthetases, initiation factors, elongation factors, and release factors
The ribosome
The platform that brings it all together and contains the catalytic center. The ribosome is a massive machine containing both RNA and many proteins. The bulk of it is RNA. In all of life, 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. The small subunit has the decoding groove through which the mRNA passes and the tRNAs read the message. 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).
Initiation factors: proteins that bring the ribosome to the message and assist in getting the machinery assembled. There are three of these in bacteria, over a dozen in eukaryotes.
Elongation factors and their partners: proteins that deliver tRNA sand move the ribosome down the message.
Termination/recyclingfactors:proteins that end the process at a stop codon,and dissociate the subunits so they can be used again..
- Be able to explain the nature of the genetic code, how it is read, and the effects of mutations to the mRNA. Name the start codon and what amino acid it encodes.
- Consists of 64 triplet codons (A, G, C, U) ; 43 = 64 different codons possible
- All codons are used in protein synthesis
- 20 amino acids
- 3 termination (stop) codons: UAA, UAG, UGA
- AUG (methionine) is the start codon (also used internally)
- Multiple codons for a single amino acid = degeneracy
- 5 amino acids are specified by the first two nucleotides only (meaning they can accept any nucleotide in the final position).
- 3 additional amino acids (Arg, Leu, and Ser) are specified by six different codons
- Describe the four phases of translation: initiation, elongation, termination, and ribosome recycling, and describe the basic elongation cycle
1. Initiation – getting the machinery assembled in the right place and thus setting the reading frame.
Initiation is the step that differs the most between bacteria and eukaryotes. However, in both the goal is the same: assemble a ribosome with the start codon (AUG) and initiator methionine tRNA in the P-site, ready to receive the next aa-tRNA in the A-site.
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.
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
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M2M 10-23-17 08-10AM Translation I-II Handout - Rissland
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.
In addition to the canonical cap-dependent process, there is a cap-independent process in eukaryotes that is driven by specific RNA sequences and structures called internal ribosome entry sites. Many viruses use IRESs to initiation translation after they shut down host cell cap-dependent synthesis. In addition, there is strong evidence that some eukaryotic mRNAs use IRESs.
Initiation is also considered to be the step at which most control/regulation of translation occurs.
- Describe the four phases of translation: initiation, elongation, termination, and ribosome recycling, and describe the basic elongation cycle.
2. Elongation–moving along them RNA and making the encoded protein.
Elongation can be though of a cycle in which each step results in one more amino acid added to the polypeptide chain. The cycle consists of an AA-tRNA entering the 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 then forms when the protein chain moves form the P-site tRNA onto the A-site tRNA. Now, the action of another 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.
-Each cycle requires 4 high-energy bonds.
- To charge tRNA: 2 (ATP-> AMP)
- To deliver aa-tRNA to A site: 1 (GTP->GDP)
- Translocation: 1 (GTP->GDP)
- Describe the four phases of translation: initiation, elongation, termination, and ribosome recycling, and describe the basic elongation cycle.
3. Termination – reading a stop codon at the end of the sequence, ending elongation, and dissociating the subunits. This is accomplished when 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.
4.Recycling: getting the ribosomal subunits ready to be used again in initiation.
- Identify the important differences between bacterial and eukaryotic translation, especially in regard to 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.
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.
In addition to the canonical cap-dependent process, there is a cap-independent process in eukaryotes that is driven by specific RNA sequences and structures called internal ribosome entry sites. Many viruses use IRESs to initiation translation after they shut down host cell cap-dependent synthesis. In addition, there is strong evidence that some eukaryotic mRNAs use IRESs.
- Explain the significance and effects of the following processes: cap-independent initiation, interferon stimulation, mRNA editing, rapamycin treatment, eIF2-alpha phosphorylation. Be able to describe when these might be important.
IRES (Internal Ribosome Entry Site) RNAs can drive a cap-independent pathway of ribosome recruitment and initiation in eukaryotes. IRESs are used by viruses, but it also appears their functional can be modulated based on the cellular conditions and thus they are probably very important for regulating gene expression.
- Explain the significance and effects of the following processes: cap-independent initiation, interferon stimulation, mRNA editing, rapamycin treatment, eIF2-alpha phosphorylation. Be able to describe when these might be important
Also, in eukaryotes, different start codons have different “strengths” depending on their Kozak context. AUGs with a weak Kozak context can be bypassed, allowing downstream AUGs to be used. The result would be two different proteins produced at certain amounts, from the same mRNA.
Explain the significance and effects of the following processes: cap-independent initiation, interferon stimulation, mRNA editing, rapamycin treatment, eIF2-alpha phosphorylation. Be able to describe when these might be important.
RNA editing – this occurs when 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
Explain the significance and effects of the following processes: cap-independent initiation, interferon stimulation, mRNA editing, rapamycin treatment, eIF2-alpha phosphorylation. Be able to describe when these might be important.
In eukaryotes, the cap binding protein (eIF4E) can be bound by 4E-binding proteins (4E-BPs) that sequester it and blocks its function. When these proteins are phosphorylated, they do not bind to 4E and this allows cap-dependent translation initiation. However, under some conditions (for example, stress), the 4E-BPs are dephosphorylated, they bind to 4E, and they block its function. This can also be induced by the drug rapamycin. This results in a shut-down of much of translation. However, note that some messages are less dependent on 4E (e.g. IRES-containing messages), and these could still be translated. The role of eIF4E levels and activity in cancer is a hot topic.
Explain the significance and effects of the following processes: cap-independent initiation, interferon stimulation, mRNA editing, rapamycin treatment, eIF2-alpha phosphorylation. Be able to describe when these might be important.
eIF2-alpha is critical for the steps that lead to binding of the initiator tRNA to the ribosome. When eIF2-alpha is phosphorylated, its activity is inhibited and this blocks initiation. eIF2-alpha can be phosphorylated by several pathways. One is induced by interferon, which is produced when a cell is infected by a virus. Hence, shutting down translation is a response to viral infection. Also, many other cellular stresses lead to phosphorylation of eIF2-alpha and so this is an important way for the cell to regulate protein synthesis during certain conditions.
Explain how bacteria can be polycistronic?
Describe how intracellular levels of iron can be regulated by translation, using this as an example of how protein-mRNA interactions regulate translation.
Identify antibiotics that operate by affecting translation
