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What is the spacing between stop codon and transcription terminator?

What is the spacing between stop codon and transcription terminator?


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When one tries to express a protein from a synthetic plasmid in E. coli, how many base pairs should there be between stop codon and the transcription terminator site?


In general, there is no need for a spacing between stop codon and terminator. They can be right next to each other.

When transcribed, transcription terminator forms a hairpin-like structure which interacts with the RNA polymerase to stop the transcription. Usually, the upstream sequence will not interact with the terminator and it will be transcribed normally. In a rare case the coding sequence might contain sequences that extend the hairpin and might result in interrupted translation. This can be avoided by doing a thermodynamical inspection of the secondary mRNA structure, but this is usually unnecessary.


Stop Codon

A stop codon is a genetic code that signals the end of protein manufacturing inside the cell, like a period at the end of a sentence. The three stop codons are nucleotide base triplets that play an important role in intracellular protein synthesis physiological and/or anatomical changes are possible if a stop codon is in the wrong position on a DNA or RNA strand, or if the code sequence is changed.


15.2 Prokaryotic Transcription

In this section, you will explore the following questions:

  • What are the steps, in order, in prokaryotic transcription?
  • How and when is transcription terminated?

Connection for AP ® Courses

During transcription, the enzyme RNA polymerase moves along the DNA template, reading nucleotides in a 3′ to 5′ direction, with U pairing with A and C with G, and assembling the mRNA transcript in a 5′ to 3′ direction. In prokaryotes, mRNA synthesis is initiated at a promoter sequence on the DNA template. Transcription continues until RNA polymerase reaches a stop or terminator sequence at the end of the gene. Termination frees the mRNA and often occurs by the formation of an mRNA hairpin.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP ® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® Exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.

Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.A Heritable information provides for continuity of life.
Essential Knowledge 3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 6.5 The student can evaluate alternative scientific explanations.
Learning Objective 3.1 The student is able to construct scientific explanations that use the structures and mechanisms of DNA and RNA to support the claim that DNA and, in some cases, that RNA are the primary sources of heritable information.

Teacher Support

Ask students to draw a timeline of the steps needed for transcription and add all the different components as specific shapes. Use different colors to label the promoter and the terminator sequences.

Review the complementarity of nitrogenous bases and the stability of base pairing as a function of number of hydrogen bonds. The couple AT/AU is much less stable than CG therefore promoter sequences will be rich in AT because it takes less energy to unzip DNA.

Ask the students, How do you recognize the beginning of a sentence? They may answer that they see a period. Answer that some abbreviations are followed by a period. So the period is not enough. Upper case is not enough either. It is the combination of period followed by a space and an upper case which indicates the beginning of a sentence. In the same way consensus sequences, which indicate a promoter region where an RNA polymerase binds, contain several elements that are required for recognition.

Use a diagram to illustrate rho-independent termination. The following drawing may clarify the text in the chapter.

Teacher Support

There can be more than one consensus sequence in a genome as there are several sigma factors that recognize different sequences. Clarify, if necessary, the role of the sigma factor and rho proteins. Students confuse transcription, termination, and stop codons.

Ask students to diagram a generic gene and label the following regions in the correct sequence in the 5'-3' direction. The regions are given in the correct order here. Change the order when giving the exercise to the class:

Sigma binding consensus sequence/TATA box
Shine Dalgarno sequence (binding to ribosome)
ATG (start codon for protein transcription)
STOP codon (polypeptide termination)
Terminator region

Students have difficulty visualizing polycistronic messages. Explain that as long as there are stop codons in the message, the polypeptides will be released and ribosomes reattached at the following Shine-Dalgarno sequence. If one were to write out the structure of a polycistronic mRNA, it would be Shine-Dalgarno-AUG-------STOP---Shine-Dalgarno—AUG-------STOP---Shine-Dalgarno—AUG---STOP.

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 2.23][APLO 3.28][APLO 4.8][APLO 4.24]

The prokaryotes, which include bacteria and archaea, are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. A bacterial chromosome is a covalently closed circle that, unlike eukaryotic chromosomes, is not organized around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid. In addition, prokaryotes often have abundant plasmids , which are shorter circular DNA molecules that may only contain one or a few genes. Plasmids can be transferred independently of the bacterial chromosome during cell division and often carry traits such as antibiotic resistance.

Transcription in prokaryotes (and in eukaryotes) requires the DNA double helix to partially unwind in the region of mRNA synthesis. The region of unwinding is called a transcription bubble. Transcription always proceeds from the same DNA strand for each gene, which is called the template strand . The mRNA product is complementary to the template strand and is almost identical to the other DNA strand, called the nontemplate strand . The only difference is that in mRNA, all of the T nucleotides are replaced with U nucleotides. In an RNA double helix, A can bind U via two hydrogen bonds, just as in A–T pairing in a DNA double helix.

The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5' mRNA nucleotide is transcribed is called the +1 site, or the initiation site . Nucleotides preceding the initiation site are given negative numbers and are designated upstream . Conversely, nucleotides following the initiation site are denoted with “+” numbering and are called downstream nucleotides.

Initiation of Transcription in Prokaryotes

Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events occurring concurrently on the same DNA template. Prokaryotic transcription often covers more than one gene and produces polycistronic mRNAs that specify more than one protein.

Our discussion here will exemplify transcription by describing this process in Escherichia coli, a well-studied bacterial species. Although some differences exist between transcription in E. coli and transcription in archaea, an understanding of E. coli transcription can be applied to virtually all bacterial species.

Prokaryotic RNA Polymerase

Prokaryotes use the same RNA polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted α, α, β, and β' comprise the polymerase core enzyme . These subunits assemble every time a gene is transcribed, and they disassemble once transcription is complete. Each subunit has a unique role the two α-subunits are necessary to assemble the polymerase on the DNA the β-subunit binds to the ribonucleoside triphosphate that will become part of the nascent “recently born” mRNA molecule and the β' binds the DNA template strand. The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Without σ, the core enzyme would transcribe from random sites and would produce mRNA molecules that specified protein gibberish. The polymerase comprised of all five subunits is called the holoenzyme .

Prokaryotic Promoters

A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription. In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among prokaryotic genomes, a few elements are conserved. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species (Figure 15.7). The -10 consensus sequence, called the -10 region, is TATAAT. The -35 sequence, TTGACA, is recognized and bound by σ. Once this interaction is made, the subunits of the core enzyme bind to the site. The A–T-rich -10 region facilitates unwinding of the DNA template, and several phosphodiester bonds are made. The transcription initiation phase ends with the production of abortive transcripts, which are polymers of approximately 10 nucleotides that are made and released.

Link to Learning

View this MolecularMovies animation to see the transcription process as it happens in the cell.


7.2C: Size Variation and ORF Contents in Genomes

  • Contributed by Boundless
  • General Microbiology at Boundless

In molecular genetics, an open reading frame (ORF) is the part of a reading frame that contains no stop codons. The transcription termination pause site is located after the ORF, beyond the translation stop codon, because if transcription were to cease before the stop codon, an incomplete protein would be made during translation.

Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop codons.

Open reading frames are used as one piece of evidence to assist in gene prediction. Long ORFs are often used, along with other evidence, to initially identify candidate protein coding regions in a DNA sequence. The presence of an ORF does not necessarily mean that the region is ever translated. For example, in a randomly generated DNA sequence with an equal percentage of each nucleotide, a stop-codon would be expected once every 21 codons. A simple gene prediction algorithm for prokaryotes might look for a start codon followed by an open reading frame that is long enough to encode a typical protein, where the codon usage of that region matches the frequency characteristic for the given organism &lsquos coding regions. Even a long open reading frame by itself is not conclusive evidence for the presence of a gene.

Figure: Open Reading Frames: Frame +1 is the ORF predicted in the database to encode a protein. +2 and +3 are the other two potential ORFs in the same strand and -1, -2, and -3 are the three potential ORFs in the antisense strand.

If a portion of a genome has been sequenced (e.g. 5&prime-ATCTAAAATGGGTGCC-3&prime), ORFs can be located by examining each of the three possible reading frames on each strand. In this sequence two out of three possible reading frames are entirely open, meaning that they do not contain a stop codon:

Possible stop codons in DNA are &ldquoTGA&rdquo, &ldquoTAA&rdquo, and &ldquoTAG&rdquo. Thus, the last reading frame in this example contains a stop codon (TAA), unlike the first two.

Bacterial genomes display variation in size, even among strains of the same species. These microorganisms have very little noncoding or repetitive DNA, as the variation in their genome size usually reflects differences in gene repertoire. Some species, particularly bacterial parasites and symbionts, have undergone massive genome reduction and simply contain a subset of the genes present in their ancestors.

However, in free-living bacteria, such gene loss cannot explain the observed disparities in genome size because ancestral genomes would have had to contain improbably large numbers of genes. Surprisingly, a substantial fraction of the difference in gene contents in free-living bacteria is due to the presence of ORFans, that is, open reading frames (ORFs) that have no known homologs and are consequently of no known function.

The high numbers of ORFans in bacterial genomes indicate that, with the exception of those species with highly reduced genomes, much of the observed diversity in gene inventories does not result from either the loss of ancestral genes or the transfer from well-characterized organisms (processes that result in a patchy distribution of orthologs but not in unique genes) or from recent duplications (which would likely yield homologs within the same or closely related genome).


Primary structure requirements for in vivo activity and bidirectional function of the transcription terminator shared by the oppositely oriented skc /rel-orf1 genes of Streptococcus equisimilis H46A

The region between the Streptococcus equisimilis streptokinase (skc) gene and the oppositely oriented rel-orf1 transcription unit contains only one termination site known to function bidirectionally in both the homologous host and in Escherichia coli. The terminator sequence is similar to other factor-independent terminators. Using two sets of point mutations that interrupt the hairpin-upstream oligo(dA) tract or the hairpin-downstream oligo(dT) tract, we examined the possible contribution of extended base pairing between the upstream rA and downstream rU residues to efficient termination and bidirectionality in both hosts, using terminator-cat reporter gene fusions in either polarity. The results show that interrupting the oligo(dA) tract preceding the hairpin has relatively little effect on terminator strength in either orientation in the homologous host, but abolishes termination in skc polarity in E. coli. Disruption of the hairpin-distal oligo(dT) tract inactivated the terminator in skc polarity in both hosts, had little effect on termination efficiency in rel-orf1 polarity in S. equisimilis, and also retained appreciable terminator activity in E. coli. In general, these alterations of the terminator sequence, together with additional mutations that reduce the spacing between the skc stop codon and the termination site or introduce a base substitution in the terminator stem, adversely affected the efficiency of termination to a greater extent in E. coli than in the homologous host. The disparity between the effects of certain mutations in the two hosts suggests that, in addition to thermodynamic properties, specific host factors, including RNA polymerase, contribute to terminator strength.

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CONCLUSIONS

WebGeSTer DB is a catalogue and presentation of intrinsic terminators. The data sets from WebGeSTer DB show that intrinsic termination is a universally conserved mechanism present in all bacterial species sequenced till date. The representative data from WebGeSTer DB are in agreement with the experimental evidence of intrinsic termination, and hence serve as a validation of the database. The database provides insight into the evolved variations in intrinsic terminators, like other successful regulatory process. The compilation would be invaluable for further experimentation on the mechanism of termination and understanding of gene expression in different bacteria.


Question: Question 1 Of 7 1 Points What Follows Is A Fill In The Blank Question With 2 Blanks. Genetic Elements Are Specific Nucleotide Sequences Located On The DNA. The Above Figures Contains A Number Of Genetic Elements That Are Involved In DNA Replication, Transcription And Translation. DNA Replication Starts At ______________ And Ends At _______________ Question .

What follows is a fill in the blank question with 2 blanks.

Genetic elements are specific nucleotide sequences located on the DNA. The above figures contains a number of genetic elements that are involved in DNA replication, transcription and translation.

DNA replication starts at ______________ and ends at _______________

What follows is a fill in the blank question with 2 blanks.

What follows is a fill in the blank question with 2 blanks.

The sections between promoter and transcription terminator on the chromosome is labelled from 1 to 5. Following transcription, which sections will be included in the resulting mRNA molecule?

  • A. section 1 to 5
  • B. section 2 to 4
  • C. section 2 and section 4 only
  • D. section 5 only
  • A. 1 section 1 to 5
  • B. 1 section 2 to 4
  • C. 2 section 2 and section 4
  • D. 3 section 2, 3 and 4

What follows is a fill in the blank question with 10 blanks.

Please identify the mRNA sequence from the below DNA fragment

3’ – ATG CCC CAC TAC GGA GTT TAG AGC ATT ACC – 5’ (Template strand)

5’ – TAC GGG GTG ATG CCT CAA ATC TCG TAA TGG – 3’ (Non-template/coding strand)


mRNA: 5'– _____________ ______________ _____________ _______________

______________ _____________ _____________. ______________. ______________

What follows is a fill in the blank question with 5 blanks.

Continue from question 6 above, please identify the resulting polypeptide sequence encoded from the above DNA fragment.

Hint: Translation only starts from AUG start codon, and ends at STOP codon. Codons must be read in the 5' to 3' direction on mRNA.

Polypeptide sequence: N' - ______________ ______________ ______________ _______________

Please put down amino acids in the 3-letter abbreviation as in codon table (e.g. put down Met instead of Methionine). The 3-letter amino acid abbreviation always starts with a capital letter followed by 2 lowercase letters.


please help me with 4. and 5. ? i am not sure on my answers.


What reading frame? - (Feb/02/2014 )

Hallo, a rookie question, but how do I know what the reading frame of a plasmid is?

I have the sequence of a plasmid. It contains a promotor sequence and after this sequence there is a restriction site and right after this restriction site there is the atg sequence. Does that mean this atg is the right start?

Or can the start codon also be one of the atg codons in the promotor?

Usually what happens is that the promoter modifies how often something is transcribed and includes a RNA polymerase binding site.  From this site, the RNA pol works its way along the sequence until it reaches an ATG site, where it will start transcription.  The promoter does not need to be in-frame with the ATG.

bob1 on Sun Feb 2 21:43:47 2014 said:

Usually what happens is that the promoter modifies how often something is transcribed and includes a RNA polymerase binding site.  From this site, the RNA pol works its way along the sequence until it reaches an ATG site, where it will start transcription.  The promoter does not need to be in-frame with the ATG.

A second question: its a plasmid that contains 3 genes in a row (so they are all , or should be, transcribed/translated as 1 gene). Now I wonder: the first ATG is the start, but there are some ATG codons in the entire gene after the first one. Will this not hinder the transcription/translation?

And what with stop codons? I did find some in the sequence, so I wonder how this can work.

The polymerase will elongate the transcript from the ATG start to the polyA signal site (usually consensus sequence AATAAA) where it detaches and adds many As to the 3' end of mRNA.

Ribosome binds somewhere before the ATG and translates codons (where ATG encodes for ordinary methinonine inside the mRNA strand and doesn't have any other function) until they reach a stop codon, after which it detaches.

If you want to translate more genes from the same ATG (so theay are polycistronic, all on the same mRNA molecule) you need to have internal ribosomal binding site (IRES) between them so the ribosome will reattach. Without it ribosome can't go over stop codon. 

Or the genes need to have their own promoter.

Alternative way of creating more proteins from one mRNA is inclusion of 2A peptide self-cleaving sequence between the genes, single protein is created (the original genes must not have the stop codon!) and then cleaved at 2A sites.

If your genes are not separated by anything and don't even have a stop codon in the middle, in that case they are 'fused', they will create single hybrid protein (and in that case I wouldn't probably call them 'genes', for clarity). You should know what exact type of plasmid you have.  

Trof on Mon Feb 3 10:32:47 2014 said:

The polymerase will elongate the transcript from the ATG start to the polyA signal site (usually consensus sequence AATAAA) where it detaches and adds many As to the 3' end of mRNA.

 

Ribosome binds somewhere before the ATG and translates codons (where ATG encodes for ordinary methinonine inside the mRNA strand and doesn't have any other function) until they reach a stop codon, after which it detaches.

 

If you want to translate more genes from the same ATG (so theay are polycistronic, all on the same mRNA molecule) you need to have internal ribosomal binding site (IRES) between them so the ribosome will reattach. Without it ribosome can't go over stop codon. 

 

Or the genes need to have their own promoter.

 

 

Alternative way of creating more proteins from one mRNA is inclusion of 2A peptide self-cleaving sequence between the genes, single protein is created (the original genes must not have the stop codon!) and then cleaved at 2A sites.

 

If your genes are not separated by anything and don't even have a stop codon in the middle, in that case they are 'fused', they will create single hybrid protein (and in that case I wouldn't probably call them 'genes', for clarity). You should know what exact type of plasmid you have.  

I think its indeed more the last one: its a plasmid with a promoter, tags for immunoprecipitation, gene of interest and fluorophore. And they are linked with glycine linkers.

So I assume its just 1 promoter for all the "genes" and 1 stop codon at the end.

But when I look look at the sequence I find multiple "stop" codons in these "genes".

an extra question: is it possible to have different reading frames in a plasmid working at the same time?

I noticed that the start codon of one of the antibiotic resistant markers uses another reading frame than the start codon of the tags for immunoprecipitation , the gene of interest and the fluorophore.

So you actually have "a gene" with a N-tag (possibly Myc tag or else) and C-tag (GFP or similar) transalted together. Glycine is just used to separate the domains.

But if you have stop codons within the sequence, it's not quite right. There are specific situations, where there is a way how to overcome stop codon, for some special purpose as I recall, but you should know if that's the case.

Where are the stop codons? Are on the expected ends of the each "gene" transcript" or somewhere in the middle?

Can you locate the polyA signal?

All transcription happens relative to polymerase bindig site (i.e. the promoter). No other relative possition is relevant. You may have several reading frames on a same DNA strand (of even on the opossite) that are off-frame to each other, but every one is "inframe" to its respective promoter. Or as was said, there just needs to be a ATG in the vicinity of the promoter and polymerase will find it, if there are more ATGs close by, probably transcription will start from either of them. But that's usually not very desired, because it has no regulation over that, so if in a living organism there are supposed to be alternate reading frames within one sequence, I would expect each of them to have it's own promoter.

its indeed a gene with an N-tag and C-tag!

I checked again for the stop codons, I can find them, but they seem to be in a different reading frame (I used the reading frame that correspondends with the ATG start codon right after the promoter).

The poly A tail, no I could not find that.

I used your sequence: AATAAA , perhaps its another one?

(I found AATAAA, but in another reading frame than the ATG start codon right after the promoter).

Trof on Mon Feb 3 12:16:44 2014 said:

So you actually have "a gene" with a N-tag (possibly Myc tag or else) and C-tag (GFP or similar) transalted together. Glycine is just used to separate the domains.

 

But if you have stop codons within the sequence, it's not quite right. There are specific situations, where there is a way how to overcome stop codon, for some special purpose as I recall, but you should know if that's the case.

 

Where are the stop codons? Are on the expected ends of the each "gene" transcript" or somewhere in the middle?

Can you locate the polyA signal?

 

 

All transcription happens relative to polymerase bindig site (i.e. the promoter). No other relative possition is relevant. You may have several reading frames on a same DNA strand (of even on the opossite) that are off-frame to each other, but every one is "inframe" to its respective promoter. Or as was said, there just needs to be a ATG in the vicinity of the promoter and polymerase will find it, if there are more ATGs close by, probably transcription will start from either of them. But that's usually not very desired, because it has no regulation over that, so if in a living organism there are supposed to be alternate reading frames within one sequence, I would expect each of them to have it's own promoter.

Poly-A signal doesn't need to be inframe. The frame only matters from ATG to stop codon, after that, there is the 3' untranslated region within which there has to be poly-A signal to physically end the template-dependent polymerization.

So if your AATAAA is after the stop codon of the C-tag, it's your poly A signal.

(you will not find "tail" in DNA, the tail is only produced on the end of the mRNA as the result of the AATAAA signal sequence and it's template-independent)

After the "genes" (the N tag, "gene" and C tag) there is a terminator (this terminator contains many stop codons . I guess this terminator is also "stop codon", I mean: the first stop codon (three letters, TAG,TAA or TGA) is to be found in this terminator sequence, not really right after the end of my "c-tag", meaning after the C-tag( the fluorophore) there are still a few 3 codons left.

Is this normal or do people normally put a stop codon (for example TAG) right after the tag sequence ? And is it normal to have a terminator sequence all the time of is the stop codon enough?

I guess it all depends on what you want?

Trof on Mon Feb 3 12:54:45 2014 said:

Poly-A signal doesn't need to be inframe. The frame only matters from ATG to stop codon, after that, there is the 3' untranslated region within which there has to be poly-A signal to physically end the template-dependent polymerization.

So if your AATAAA is after the stop codon of the C-tag, it's your poly A signal.

(you will not find "tail" in DNA, the tail is only produced on the end of the mRNA as the result of the AATAAA signal sequence and it's template-independent)

For ribosome, only one and first stop codon matters. 

Imagine you're reading a book, until you get to a command saying "now stop reading and go away". And you do. So eventhough there may be furter text in the book, of furter commands saying you to do something, it doesn't matter, because you already stopped and left. That's what ribosome does.

The sequence after THE stop codon is kind of "junk" (in fact it most likely is NOT useless in the organisms, beacuse it may contain regulatory sequences, that may affect translation rate or bind other factors doing various things), but it's not important for he ribosomal elongation of the protein.

 Terminator contains stop codon (possibly for every frame there is, just to be sure), poly A signal for the respective type of organism (prokaryotic, eukaryotic). Some may contain expression enhancers. This is just a common building brick of plasmids.

As for the C-tags, there doesn't need to be end exactly after the functional protein domain. There can be some "dummy" amino acids, if they don't affect the function of the tag, they doesn't matter. 


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    CONCLUDING REMARKS

    Here, the universal U6 promotor-based system overexpressing various human endogenous tRNA iso-decoders was developed. Using several experimental set-ups we demonstrated the following. i) Yeast and humans share at least two rti-tRNAs (Trp and Tyr) as dosage-dependent enhancers of readthrough on UGA or UAG and UAA stop codons, respectively. (ii) The ‘tRNA score’ is a reliable predictor of a functionality of a given tRNA iso-decoder in translation. (iii) Overexpressing tryptophan tRNA: (a) boosts SC-RT on reporters bearing SCCs of mRNAs encoding cellular and viral genes known to be SC-RT subjects, and (b) enhances restoration of a functional p53 protein production from its mRNA containing mutations converting codons cognate to tRNA Trp to nonsense PTCs. Therefore, we propose that tissue-to-tissue specific levels of selected readthrough-inducing tRNAs might have a significant impact on the balanced expression of the parent versus extended forms of cellular, as well as viral proteins. As such, we envisage that a cell-type specific modulation of the rti-tRNA levels could help to combat human pathogens whose life cycle relies on SC-RT and, perhaps, also support the developing RTID-driven treatments of PTC-caused diseases.



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