Biochemistry 3380G Final: Topic 24 - Translation.docx

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8 Feb 2015

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Overlap would place significant restrictions on what amino acid other. 3 stop codons: uaa uag uga, one start codon, aug. Helps maintain function of proteins should a mutation occur: universal, the code has evolved once. Dramatic effect unless at the end of the protein trna: serve as the vehicle that brings the amino acid to the growing polypeptide chain, similar structure for all trnas, stem-loop structure resulting from internal base pairing. In them there are nonconventional bases: they are not inserted as nonconventional they are altered post transcriptionally. 2 key single stranded regions 3" acceptor site (site of amino acid attachment) and anticodon which base pairs with the codon: anticodon: key in codon recognition, base pairs with the codon normal base pairing rules apply. 3" acceptor: coupled in a high energy ester linkage to a specific amino acid. In 3d they look more like an inverted l.

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4. When a scientist makes a genetically modified bacterium as a tool for other experiments, there is always a fear that it might get out of the lab and wreck the environment. So there has been an interest in developing a âkill switch,â by which the scientist could ensure the bacterium only grows in the lab. Recently, one way to do this has been developed, wherein an artificial amino acid (that is not found in nature) is required for bacterial growth. To make this work, the artificial amino acid (call it âamacXâ) must be encoded by the bacterial mRNA, recognized by a tRNA, and must be provided as a growth factor in laboratory media.

A. Look at the Genetic Code. How would you decide which of the codons you should use to represent amacX? (There may be more than one possible choice, so focus more on general principles than on a specific codon.) Are there any codons that you couldnât use for this purpose? Why couldnât you use them?

B. The gene for which enzyme would have to be mutated to make this work? Specifically, what mutation would need to be introduced to this enzyme? [Make a sketch as part of your answer.]

C. Letâs say this organism escapes into a natural environment where there is no amacX. Make another sketch showing what a Ribosome from this bacterium would look like when it encounters an amacX codon during translation. What would happen to this Ribosome?

magentaporpoise625

I need help with Part B. I believe that part A is correct.

a) You also wish to synthesize a degenerate oligonucleotide primer that could be used eventually to clone DNA that encodes your mutant protein. Design this degenerate oligo, keeping in mind the rules we talked about in class. More information about designing degenerate primers and primers in general are located on the following page.

Write out the sequence of this oligo here based on this amino acid sequence (Met Ala Pro Met Val Ala Glu):

My answer (Is it correct?)

I got:

5â AUG - (GCU/C/A/G) - (CCU/C/A/G) - (AUG) - (GUU/C/A/G) - (GCU/C/A/G) - (GAA/G) â3

3â UAC - (CGA/G/U/C) - (GGA/G/U/C) - (UAC) - (CAA/G/U/C) - (CGA/G/U/C) - (CUU/C) 5â

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(What I actually need help with and have no idea where to begin. Please help me)

b) If you synthesized the above oligonucleotide primer, how many different oligos would be present in the mixture?

Follow these rules Modified from the Thermo Fisher web site:

Designing degenerate primers:

Write out your amino acid sequence, label amino and carboxy termini

For each amino acid, use the genetic code to predict the various nucleic acid codons that can encode this amino acid. For all but Met, you should have redundancy present.

Now you need think about 5â and 3â considerations, and keep in mind that DNA within genes is double-stranded. It is helpful to keep in mind that the 5â end of a gene corresponds to the amino terminus of a protein. Using 5â and 3â labels write out a single strand DNA sequence corresponding to the your amino seq of interest. You may choose to start with only possible codon for each amino acid, or you can do all possibilities using the notation I showed you in class to account for the wobble position of codons.

Now make your DNA double stranded by filling in the opposite DNA strand; label the 5â and 3â ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.

If you havenât already, now consider the redundancy present on both strands, and make sure you have indicated sequences that account for all possible redundancies.

Realize that when a researched synthesizes a degenerate oligo that the final synthesis contains oligos with every substitution possible. The amount of degeneracy is defined by the number of different primer combinations in the mix. You can determine the degree of degeneracy by multiplying the number of changes present at each position together. If a position has no degeneracy then you multiple by 1 for that position.

Keep in mind that the trade-off between primer specificity and efficiency can be modified by altering the degeneracy of the primer. For example, the more degenerate the primers, the less specific annealing will be; however, decreased degeneracy will allow more potential to identify unknown variants. Anything over 1024 fold degeneracy is at the upper limit of potential usefulness.

Biochemistry 3380G Final: Topic 24 - Translation.docx

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