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14

Th reason for this is that for the third base of the tRNA non-Watson-Crick pairing is allowed. This phenomenon is called "Wobble base pairing". See the figure (from here) for illustration (from here): If you have a look at the codon table for amino acids, than the variation in the code for one amino acid mostly happens on the third position (from here): ...


8

A pre-tRNA is transcribed from tRNA genes in DNA by RNA polymerase III. Processing occurs in the nucleus, where a 5' sequence is cleaved by RNase P, the 3's CCA motif is added, and ~10% of the nucleotides are substituted. The tRNA are transported out via the pore complexes. Aminoacyl-tRNA synthetase enzymes attach amino acids in the cytoplasm in a 2-step ...


7

No, this will not happen. mRNAs are inspected in the nucleus before they are exported into the cytoplasm (at least in eukaryotes), where transcription and translation don't happen at the same place. This ensures that no mRNAs without stop codons or premature stop codons are exported. This phenomenon is called "mRNA surveillance". mRNAs that do not pass this ...


6

A clarification on introns and exons. While it is true that introns are not a part of the mRNA as March Ho said, they are essentially transcribed. This may seem trivial but it is important to note. So: Both introns and exons arise from the transcribed region Exons need not necessarily form the ORF (i.e. be translated to proteins) Regarding intronic ...


5

I think it is relatively unlikely that the RNA will degrade under this conditions. For the future, I would handle this differently: You can centrifuge the cells and snap freeze them in an appropriate buffer in liquid nitrogen and then store them at -80°C. I would not freeze the dry pellet, as these are often hard to re-dissolve after freezing. ...


5

Start and stop codons are instructions for the ribosome to start and stop protein synthesis, respectively. The region between the start and stop codon (inclusive of them) is called ORF (open reading frame) or sometimes CDS (Coding sequence). Why does ribosome need explicit instructions for start and stop? Ribosome recognizes an RNA as a mRNA if it has ...


5

This question can't be answered with a simple yes/no, but I would say that the analogy of DNA being the "code" used by cells is a reasonable one, if taken with a number of other considerations. DNA function When Watson and Crick first described the structure of DNA (being a double-stranded sequence of the nucleotides Adenine, Cytosine, Guanine and ...


5

Not very common, and not found so far in nature, but they exist and are called deoxyribozymes. Additional information: Deoxyribozymes are the equivalent of ribozymes in the DNA world and can function as catalysts for different biochemical reactions, such as DNA cleavage. While DNAzymes (short name) were synthesized in a laboratory context (In-vitro) and ...


4

Short answer: Yes, you can. Usually these are coupled systems for transcription and translation. For these you clone the gene of interest into a vector which contains a prokaryotic promoter which is then used to generate the mRNA in the tube. This mRNA is then translated in the second step into a protein. This works very well, but the protein is missing ...


4

Watson-Crick base pairing can be violated by wobble base pairing. The 5' of the anticodon has more freedom in binding, that is why, for many amino acids, the last part of the codon has more possible characters.


4

As far as I know this has never been thoroughly analyzed for RNA, but there is an excellent paper on the precipitation of DNA and the usual conditions in BRL Focus by Zeugin (see below for the paper). Since DNA and RNA are pretty much the same (except for one OH-Group) and the conditions used for precipitation are also similar, I think we can use this for a ...


4

According to my book Molecular Biology Principles of Genome Function by Craig et al, eukaryotic RNA degradation does not have an initiation, as in bacteria where pyrophoasphate hydrolase hydrolyses the 5'-triphosphate to 5'-monophosphate, and is therefore the initiatior. In eukaryotes the polyA-tail is shortened by a deadenylase enzyme complex which ...


4

Qiagen and Life Technologies call it RNA content, total RNA, or total cellular RNA.


4

It sounds like you have considered most of the obvious alternatives (and thank you for clarifying the question). I suppose the first question in an alternatively spliced transcript with a retained intron is whether the open reading frame of the protein is maintained. If there is a termination codon that now becomes in-frame due to the intron then the ...


4

One can prepare rabbit ribosomes from reticulocytes (immature red blood cells), and by providing a few cofactors required for an active extract, create an in vitro system that will translate virtually any eukaryotic mRNA that contains the correct sequence determinants. You can purify mRNA from your cells or tissues of choice, or you can synthesize an mRNA in ...


4

While Ankur's answer is correct, it must be noted that not all non-coding RNAs are introns. An intron must be excised from an mRNA, which therefore means that any non-coding RNA that is not part of an mRNA cannot be an intron. For example, rRNA and tRNA are all examples of non-coding RNAs that are not introns, since they are not part of mRNA. miRNA may ...


3

Comparing Biopython MetlingTemp to other calculators. I have written the recent version of MeltingTemp in Biopython's SeqUtils. I have extensively tested the Tm calculations against other programs like MELTING and Primer3Plus and other online Tm calculators with consistent results, thus I'm pretty confident that there is no gross error in the module. The ...


3

I'm assuming that you have a solution of bacteria in your buffer directly after growing them for some time, and without performing a lysis step yet. The 2% SDS step looks like it is for lysis of your cells. In that case, centrifuging the bacteria and resuspending them in lysis buffer is a very common step in many protocols. Separating whole bacteria from ...


3

21joanna12, look into snRNPs. These are parts of the splicosomal apparatus and some of them (the U1 and U2, U11 and U12 snRNPs) are also the guideposts that bind near the splice junctions at the end of introns. These help guide the splicing apparatus to the splice sites. There are also proteins that bind RNA and interact with the splicing apparatus to ...


3

Yes, transcription of a polyA sequence will be unreliable: for a repetition of 15 times the same nucleotide, the rate of transcription errors will be huge. For example in E. coli, this paper http://www.ncbi.nlm.nih.gov/pmc/articles/PMC331007/ gives some order of magnitude of transcription errors caused by repeats of nucleotides. According to their data, for ...


3

It is important to have start and stop codons so that the molecular machinery of the cell (ribosome etc.) "know" where the actual transcript starts and ends. This is especially important, since mature mRNA contains untranslated regions which are of regulatory importance. These regions occur on both sides of the coding sequence called 5' and 3'UTR ...


3

Not all the RNA is to be translated into proteins. Actually most of it is for regulation and sometimes unknown use. There are non coding regions before the start codon and after the stop codon. Hence the need for both.


2

Next to helping to define the region for translation on the mRNA, the stop codon also prevents transcripts that have been frame-shifted by a mutation from translating into big proteins, and also helps flagging the mRNA for destruction (nonsense-mediated RNA decay). This is because in random sequence, a codon has 3/64 chance of being a stop codon, so in a ...


2

I don't think bleach can denature RNA. Bleach is an oxidizing agent and it will damage the RNA. Also, the protocol mentions addition of hypochlorite before heating which I think is illogical because heat will decompose it. For testing RNA integrity you need not make a denaturing gel. What you can instead do is to heat the RNA with the 2xRNA loading buffer ...


2

In my opinion you should try low input library prep protocols because in my experience even with a decent quantity of RNA, standardizing the sequencing is not an easy job. The manual says that this kit works with 10-20ng of RNA; always assume the higher limit to be true. There is a higher chance of loss (relative) during extraction also. Try doing ...


2

MicroRNA (miRNA) are gene-regulatory RNAs that are loaded onto the RNA-induced silencing complex (RISC) and interact with partially-complementary targets on mRNA to suppress protein expression. The miRNA is originally double-stranded and composed of strands about 21 nucleotides long; on loading onto RISC, one strand is degraded and the other, the "guide" ...


2

Some general aspects about miRNA nomenclature in addition to canadianer's answer: mir refers to the pre-miRNA whereas miR refers to the mature form mir-x-y mir-x-z for example mir-1-1 and mir-1-2 refer to different precursors that give rise to same mature miRNA (i.e. miR-1). These different precursors can be regulated differently. The mature miRNA ...


2

Actually, whether polyadenylation protects an mRNA or makes it susceptible to degradation depends on the organism. From Dreyfus and Régnier 2002: In eukaryotes, poly(A) tails usually act as stabilizers of intact mRNAs, whereas in E. coli they serve to accelerate the destruction of fragments. This is due to different protection mechanisms in prokaryotes ...


2

As to why they don't recognize RNA-DNA heteroduplexes (which are present during transcription, for example), I suspect that the methylation which protects bacterial genomic dsDNA (see the DNA modifying enzyme section of this Columbia University lecture for more info) also protects RNA-DNA hybrids, as the genomic DNA would still be methylated. Note that most ...


2

This paper found that these enzymes recognize RNA:DNA heteroduplexes. Such duplexes are unlikely to be encountered in vivo. They are present when DNA is primed for replication, but these duplexes are relatively short and thus are less likely to randomly contain a recognition sequence. Furthermore, if the recognition sequence is found in the primer, the gDNA ...



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