[Nescafe]

Hi,

I recently had a post here about plasmids where I was wondering whether they are single or double stranded and the answer which I agreed on was that it is double stranded. I was recently doing some reading on incorporation of unnatural amino acids and came across a slide where they showed a single stranded plasmid with TAG. (http://www.scs.illinois.edu/silverman/chem590cdocs/Lecture12.pdf) It is on page 9 of 17 or slide 12-18 (slide # is on the top right of each slide).

I understand that the code for TAG on mRNA is UAG, but does this mean that first the plasmid is complemented with another strand of DNA that matches TAG with ATC and then ATC is transcribed to mRNA in which the matching code for ATC is UAG?

Am I missing something cause I am confused :S

Nescafe.

PS I understand that the slide I am referring to is not necessarily concerned with my question. I am just curious as to why they say/show/draw TAG -> UAG. Is it because we start with single stranded circular (plasmid) DNA then make double stranded at which point we have TAG -> ATC, which transcribes gives UAG? I just repeated my question in my PS

PPS Sorry for repeating my question in my PS =P

[Arkcon]

Yes, the double stranded plasmid DNA complement to the sequence TAG is ATC.  If the strand with the TAG sequence is being transcribed to mRNA then it will be AUG, unless, as the slide points out TAG is a Stop codon.  According to the context of that slide, whether TAG is transcribed or whether its a Stop depends on the mRNA, if its a mutation or not.  Alternatively, you could ligate another gene into that Stop codon, ruining the protein it codes for.  If that protein is important, say antibiotic resistance, you can now select against that colony.

[Babcock_Hall]

I don't have time for a lengthy answer today, but just two quick observations.  Some plasmids are single stranded, at least for part of the time.  Plasmids based on M13, a bacteriophage, come to mind as something which can be encountered in both forms.  My knowledge of unnatural amino acid technology is limited, but I think that they used to be based on nonsense suppressor tRNAs.  One of the slides alludes to the Amber stop codon.

http://www.bayoubiolabs.com/biochemicat/vectors/pUCM13/

[Yggdrasil]

Here's how site specific incorporations of unnatural amino acids (UAAs) works: 

1) You take a plasmid containing your gene of interest and find the codon corresponding to the amino acid that you want to mutate.

2) You change that codon to a TAG sequence on the coding strand (see the my post in the thread on site-directed mutagenesis for how to do this).  This results in TAG on the coding strand of the DNA, CTA on the non-coding strand, and UAG on the corresponding mRNA.

3) You introduce this plasmid into a special strain of E. coli that has been engineered to allow for UAA incorporation.  This strain will have three important characteristics:

A)  UAG is normally a stop codon that would terminate protein synthesis, producing a truncated protein.  However, there are strains of E. coli known as nonsense suppressor strains that do not recognize certain stop codons correctly.  We choose the UAG stop codon because, of the three stop codons, it appears least frequently in the E. coli genome.

B)  The strain will also express an enginneered tRNA that contains a CUA sequence on its anticodon loop.  This tRNA will bind to the UAG codon on the mRNA to incorporate the UAA into the protein during ribosomal protein synthesis.

C)  Finally, the strain will also have an engineered aminoacyl tRNA synthetase that will attach the UAA to the engineered tRNA.

[Yggdrasil]

Yes, the double stranded plasmid DNA complement to the sequence TAG is ATC.  If the strand with the TAG sequence is being transcribed to mRNA then it will be AUG, unless, as the slide points out TAG is a Stop codon.  According to the context of that slide, whether TAG is transcribed or whether its a Stop depends on the mRNA, if its a mutation or not.  Alternatively, you could ligate another gene into that Stop codon, ruining the protein it codes for.  If that protein is important, say antibiotic resistance, you can now select against that colony.

Note that stop codons control the termination of translation (protein synthesis) not transcription (mRNA synthesis).  Introduction of stop codons into mRNA will not stop transcription.

Also, the DNA complement of 5'-TAG-3' is 5'-CTA-3'.  Remember that paired DNA strands run in opposite directions and by convention we almost always write DNA sequences 5'->3'.

[Arkcon]

Gah.  And I also said that it depened on mRNA mutation, which isn't what the slide said, but instead on a mutation in the aminoacyl-tRNA synthetase.  Too early in the morning for me to be reading pdf's.

[Babcock_Hall]

Just to clarify my comments about phage M13, it is single stranded at some points, but its replicative form (RF) is double stranded.  Kornberg's book "DNA replication" deals with this starting on page 479.

[Nescafe]

Thank you so much for the posts, very helpful. So if the template (non-coding) DNA sequence has the 5`-CTA-3`, then the mRNA is synthesized complementary and antiparallel to this template strand (anticodons) of DNA giving us 5`-UAG-3` on the mRNA. The resulting mRNA (5`-UAG-3`) then consists of codons corresponding to those in the coding strand of DNA (5`-TAG-3`).

If what I said, is confusing yet truem then it means that I made sense of what you said, if not, then I suck =(

Nescafe.

[Yggdrasil]

Yes that is correct.  That definitely is a point of confusion for many students.  Even though we generally say that one strand of DNA (the coding strand) contains the information from the gene, RNA polymerase actually reads from the opposite strand (the non-coding/template strand) in order to synthesize the mRNA.

Also, in the pdf, even though someone may draw a plasmid as a circle containing only one line, that usually does not mean that it is single-stranded.  Very often, diagrams will take for granted that DNA is double-stranded and just draw it as a single line not representing the two strands separately.

[Nescafe]

Yes that is correct.  That definitely is a point of confusion for many students.  Even though we generally say that one strand of DNA (the coding strand) contains the information from the gene, RNA polymerase actually reads from the opposite strand (the non-coding/template strand) in order to synthesize the mRNA.

Also, in the pdf, even though someone may draw a plasmid as a circle containing only one line, that usually does not mean that it is single-stranded.  Very often, diagrams will take for granted that DNA is double-stranded and just draw it as a single line not representing the two strands separately.

That you SO much, very helpful!

Nescafe.