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35

Answering my own question after reading the 2018 Nature review article “mRNA vaccines — a new era in vaccinology” The resources and motivation engendered by the COVID-19 pandemic are a major factor in the development of the first mRNA vaccines approved by national governments. However, before the COVID-19 pandemic, there were recent advances in mRNA vaccine ...


12

A good question (if a little mixed up on transcription vs. translation!) AUG is not always the start codon, but whatever the codon is it will always code for Methionine (or fMet, but still a variation on Met), even if the codon codes for a different amino acid otherwise. A separate transfer RNA (tRNAi, the initiator tRNA) is used for the arrangement of ...


11

See this paper. They have studied RBP-protected sites in the entire human transcriptome by RNA-protein crosslinking followed by RNAse digestion and sequencing: PIPseq. Figure 1 of the paper shows distribution of protein protected sites in RNAs. They also correlate it with different regions of mRNA and its expression. They show number of protein protected ...


11

They're both correct. The confusion stems from the book talking about the anticoding strand as well as the newly-formed coding RNA strand, whereas Khan Academy talks only about the coding strand. From the book: The anticoding strand is transcribed from 3' to 5'. Therefore the coding strand is produced from 5' to 3', meaning the very first nucleotide ...


10

Ribosomes don't read DNA. Transcription adds several layers of regulation tactics: you can control transcription itself, you can control mRNA. mRNA amplifies genetic information that you need at the moment. Having many copies of a matrix really helps. Some prokaryotes have splicing. It's not a good idea to splice your DNA. EDIT per your request of some ...


8

I would strongly recommend looking in more detail into available resources for SD and Kozak sequences, wikipedia basically answers these questions and has plenty of further reading if you desire to explore these questions. At the same time, remember that these are statistical processes involving thousands of molecules, rather than deterministic processes ...


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 ...


7

Transcription occurs in a special structure known as transcription bubble. Inside the bubble are present the mRNA, template DNA being transcribed and the RNA Polymerase. Upstream of bubble is the DNA already transcribed and downstream is the DNA to be transcribed. There is not enough space in cell to have completely unfolded DNA for transcription, so ...


7

@Thymine's answer is correct. I just thought I'd post a more graphic answer for clarity. <==(RNA Pol)3'------------------------- 5' 5' ------------------------------------------------------ 3' 3' ------------------------------------------------------ 5' The RNA Polymerase is synthesizing on the 3' to 5' strand, but nucleic acids are ...


6

There are some tools for predicting the binding: TargetScan (based on seed match [primary], extra pairing, sequence context 1 — nucleotide composition around the site etc [secondary]) miRanda (based on hybridization stability and seed match[primary] and sequence context [secondary]) PicTar (adds a layer of evolutionary conservation criteria) 1 Context ...


6

Yes, you can find mutations in the genomic DNA which affect splice acceptor sites. Wikipedia lists the following outcome: Mutation of a splice site resulting in loss of function of that site. Results in exposure of a premature stop codon, loss of an exon, or inclusion of an intron. Mutation of a splice site reducing specificity. May result in variation in ...


6

The two stop codons are obviously to prevent read-through of the termination codon. Why this should be necessary is not clear to me, but the following may be relevant: The synthetic mRNA differs from the natural mRNA in a particular respect that is easier to explain with reference to the transcript map of the virus, below. The two ORFs 1a and 1b are ...


5

First you have to define what you really mean by miRNA-X targets mRNA-Y. If you mean direct targeting then there are assays to verify it. To verify if the miRNA can potentially target the mRNA independently, what is routinely done is a reporter downregulation assay (usually luciferase). You clone the 3'UTR of your target mRNA downstream of the reporter gene ...


5

It is very difficult to answer this one for certain but I think the codon wheel representation is sometimes attributed to Rosemarie Swanson who represented the amino acid code using Gray code (or reflected binary code). Swanson's work actually went quite a bit deeper than the simplified codon wheels you find in ordinary textbooks (ordering by size of the ...


5

I don't think definite answers exist but I can think of theoretical reasons: First, Poly A tail can efficiently be synthesized because ATP is the most readily available nTP, being the main energy source. More to the mechanism, but this is no explanation to the evolution: The termination sequence 'TTTATT' on the DNA template and 'AAUAAA'on the RNA is the ...


4

The only information you are missing is a way to identify the splice sites. There are many ways of doing what you need. The simplest, assuming you are sure of the origins of the mRNA, is to use a BLAST flavor, either plain BLASTn or, even better, BLAT, to compare your mRNA sequence to the genome of interest. BLAT really should be all you need if the mRNA ...


4

From recent empirical research (Wang et al., 2018) on eukaryotes, basically the non-AUG start codons have context-dependent [translation initiation] efficiency, while AUG is a "sure thing", i.e. the nucleotides surrounding it have little impact on its efficiency. There are some theoretical biochem explanations for this, which I'll just quote as-is: We ...


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

Yes you are correct. These mRNAs, that lack stop codon will cause translation to continue into the poly-A tail (it will result in addition of lysines not phenylalanine). Since no stop codon is present, the ribosome remains attached to the mRNA. Under these circumstances, a pathway known as non-stop decay is activated. An important protein in this pathway — ...


4

mRNA specific queries Add this to your NCBI query: AND mrna[filter] If it's not available in major databases it may not be known. However mRNA data can be obscure to find even in those major databases but it should be there. I would be surprised if that is the case, however since you're not working with model organisms there may well be nothing available. ...


4

Concise Answer The 5′-UTR region of a eukaryotic mRNA is derived from the RNA transcript of the region of a gene between the transcription start site and the DNA corresponding to the translational initiation codon. It differs from that region of the initial transcript in most cases by having a modified guanosine nucleotide added at the 5′-end in a ‘cap’ ...


4

According to this Wikipedia article: "The Shine-Dalgarno (SD) sequence is a ribosomal binding site in bacterial and archaeal messenger RNA, generally located around 8 bases upstream of the start codon AUG.1 The RNA sequence helps recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis by aligning the ribosome with the ...


4

What is Protein Expression Level? This was the original title of the post, which I edited myself because I regard the answer as trivial, but the question as more substantial. To deal with the trivial first: ‘Level’ is not a scientific unit, and can only be used unambiguously as a scientific term in its English sense in relation to liquids, e.g. “The level ...


4

This is the temperature at which RNA is stored for long term storage without the occurance of degradation. It is also standard lab routine to store RNA at this temperature. Conviniently, this temperature can be reached by dry ice (-78°C), which makes shipping a lot easier. However, there are indications by Biontech themselves that the vaccine will be stable ...


4

I am asking this question to understand whether the cells which are used to create spike proteins are attacked by the immune system. Yes, that is the aim of RNA vaccinations! But don't worry, that's a good thing, as can be concluded from this review-paper, that emphasizes the advantage of RNA vaccinations to invoke cellular immune response: Although ...


4

The mRNA won't be entirely perfect, but the imperfections will not matter. The spike protein is big, encoded by thousands of base pairs in the virus. In the virus itself, it's not entirely homogeneous: there are always going to be small mutations that cause different variants to be produced, but nearly all of those are nearly identical. The mRNA in a vaccine ...


4

If I understand your question and graph correctly, your Y-axis is log(x/REF), where REF is some external standard. Your "Ref" on the x-axis you expect to be the same as REF, so that log(Ref/REF) "should be" zero, but you find it is not. However, it looks like the mean(log(Ref/REF)) is still approximately zero. This is what I would expect: ...


3

The protocol you are using will not only leave the sample with rRNA but also non coding RNA. Many RNA protocols will separate mRNA by affinity of a carrier to the polyA tail. This protocol references an older paper that estimates that only 5% of RNA is mRNA. I'd be surprised if this ratio changed by more than 2-3 fold in drosophila. I assume that %age ...


3

Shigeta's got a point: the ribosome is latched onto the mRNA so those two are intrinsically linked. You're really asking whether the ribosome comes off first or whether the tRNA does, but it's actually the new polypeptide, which makes sense: The stop codon is recognized by a protein, the polypeptide chain release factor (RF), which triggers the ...


3

First of all, it is the coding sequence, the open reading frame (ORF), and not the gene that starts with an AUG. Also, there are actually quite a few ORFs that start with different initiation codons, they are just the exceptions rather than the norm. As for the need, you can think of START and STOP codons as punctuation. The AUG is read like the first (...


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