16

In my opinion, Prof. Allen Gathman's "great 10-minutes video on Youtube" is a pretty waste of time if you already know how hydrolysis happens. In fact, he has not considered the 3'->5' route in an unbiased manner; he doesn't seem to look at the possibility of a triphosphate appearing at the growing 5' tip of the strand in the 3'->5' case. Actually, the ...


14

The RNA world hypothesis states that self-replicating RNA (that is, an autocatalytic RNA polymerase) was the first form or precursor of life. So, in that context, your question is basically asking how life originated. The obvious answer is that we don't know (currently anyways), but I'm going to take this opportunity to describe a few really neat experiments ...


9

Short Answer In a nutshell, DNA sequencing technology has a limit to how long a stretch of DNA it can read in one go. Long Answer So what most commonly occurs is the length of DNA you wish to sequence needs to be (almost randomly) chopped up into given lengths (depending on the technology) and each length or read is sequenced in parallel. But now you don'...


8

The pattern we see in B. subtilis is quite common in prokaryotes. The origin of replication is shown at the top of the genome diagram. DNA replication proceeds bidirectionally from this point. In the B. subtilis diagram, most genes are located along the leading strand in each direction. Even in E. coli, by the way, important genes, including all rRNAs, tend ...


8

Take a look at this schematic of a mature mRNA. [source] The coding region (ie the part that is translated) is between the start and stop codons, but the 5' and 3' untranslated regions (UTRs) are also transcribed by RNA polymerase; these are part of the first and last exons, respectively. The transcription start site is labelled right in front of the 5' ...


6

Actually there is a polymerase that catalyzes 3' - 5' elongation. See for example the Thg1 superfamily. "Doing it in reverse: 3'-to-5' polymerization by the Thg1 superfamily." Jackman, et al.


5

You mix up translation and transcription. Transcription creates mRNA from DNA template. Transcription also includes splicing, that is excision of introns so that mature mRNA contains only exons. In your example it goes like that: DNA (chromosome): ---A----B--...--Dstop---E--- premature mRNA: A----B---...---Dstop--E---polyA mature mRNA: AB..DstopE-polyA ...


5

Summary: In bacteria or organisms with only one well defined replication origin and a circular chromosome, yes for a given DNA region the same strand is replicated discontinuously. In high order animals, which replicate chromosomes using several origins of replication (ori), this is less clear as the way ori are recognized is still not fully understood but ...


5

No, this does not happen, as the DNA polymerases used for PCR are DNA dependent. This means that they only synthesize DNA when it is bound to DNA. Even if your primers bind to the RNA, the polymerase will not starting new strands here. To use RNA as a template for PCR you first need to reverse transcribe it.


5

In the (beautifully rendered) video you linked to, the green molecules are DNA polymerases. So you can already see that there are more than two DNA polymerases at work! At each replication fork, there is generally one DNA polymerase working on the leading strand, but on the lagging strand, multiple DNA polymerases may be working at the same time (as ...


5

I wouldn't really say RNA polymerase is "creating" the hydrogen bonds so much as it's thermodynamics that creates them. When we talk about an enzyme "creating" a bond, what we're generally referring to is an enzyme facilitating a reaction by lowering its activation energy so that it can proceed. However, in the hydrogen bonding between base pairs, there's ...


4

Well remember that an mRNA, the RNA that is destined for the ribosome, has already been processed by the time it gets there. This is done by the splicing machinery, among other things (capping enzymes, for example). But the splicing machinery isn't smart in itself. Rather, the gene has the elements, just like it does to select the RNA Pol it needs, to direct ...


4

This paper describes an allele-specific PCR protocol to detect CAG repeat expansion in MJD patients: Maciel P, Costa MDC, Ferro A, et al. Improvement in the Molecular Diagnosis of Machado-Joseph Disease. Arch Neurol. 2001;58(11):1821–1827.


3

Transcription always proceeds in the direction 5' (5-prime) to 3' (3-prime) on the coding strand of DNA. Binding of both transcription factors and RNA polymerase to DNA depends on sequence motifs in the DNA. Transcription always happens in the same direction with respect to the chemical structure of the coding DNA strand, while the transcription direction ...


3

Deep sequencing is naturally error prone. Sequencing will never be perfect, because no enzyme will ever perform 100.00000% perfectly. In Illumina sequencing, you put your starting molecule down on the flowcell, then the polymerase makes a cluster of copies around it. But at each step of building each copy, there's a chance the polymerase will make a ...


3

Cell division rates may exceed DNA replication rates, because DNA replication can occur in parallel, while cell division is always serial. In a circular chromosome like E. coli, replication starts at the origin of replication. Once the origin is replicated, another chromosome replication can be initiated there, while the former isn't even finished. This ...


3

Though Escherichia coli has only one origin of replication (oriC), replication proceeds in both directions from it. Thus, based on your calculations, DNA replication takes 40 minutes (which is still too long if the doubling time is 30 minutes). The solution is to start replication again before the previous round has completed, and here's some research which ...


3

What makes you think that the lac repressor will still be bound at the target gene? In strains with this type of regulatory configuration (usually with DE3 in the genotype) the idea is that IPTG induction turns on expression of T7 polymerase from a lac promoter on the chromosome, and then this T7 polymerase then transcribes the target gene on the plasmid. ...


3

It appears that this question is one of terminology, so I am answering it as such. Convention for representing features in DNA sequences The convention is that in indicating any sequence feature† in a protein-coding gene on double-stranded DNA, a single strand‡ is represented — the one from which the amino sequence could be read using the genetic code (...


3

Except for the TATA-binding protein (TBP), there are no common transcription factors for each RNA polymerase. However, there are homologies between many of the factors used by the different enzymes. The paper Conservation between the RNA Polymerase I, II, and III Transcription Initiation Machineriesa gives a good overview over the different initiation ...


3

Non-templated terminal addition by certain DNA polymerases is apparently dependent on the base stacking between the incoming base and the existing duplex (Fiala et al., 2007*). This was verified by using a non-base-pairing (but better stacking) nucleotide analogue called deoxyribo pyrene nucleoside triphosphate (dPTP). Adenine seems to have a higher ...


3

Although the answer provided by @StephenB is essentially correct, I’d like to supplement this with more visual illustrations — including a link to an animation which the poster requested — as well as reiterating the important points he makes about enzymes. This is a question about enzymology and structural biology and is essentially chemical in nature. ...


2

The polymerase used in PCR is extremely stable at room temperature. I would expect you to be able to continue your PCR reaction from the cycle it was stopped at without too much trouble. However, it is important to not over cycle your product. This will produce non-specific products. If a clean result is imperative, take a 1-5ul sample from your original ...


2

In short: Nucleotide-associated proteins are DNA-binding proteins that bind DNA. RNA-polymerase associated proteins bind RNA-polymerases and are required for its functionality. In more detail: DNA-binding protein is a higher level term that comprises all proteins that bind to DNA. These can be seen as nucleotide-associated proteins as they interact with ...


1

The beauty of RNA polymerase II is its faculty to act like a sensor towards damage to genetic data. The response of the RNA molecule is variable on the type of DNA lesion which presents itself. DNA lesions may be a consequence of ultraviolet radiation, or through the metabolic by-products of oxidative free radicals. These specific faults within the lesion, ...


1

In E. coli most RNA polymerase molecules are bound to DNA, and those that aren't are within easy reach of the DNA. According to Larry Moran A typical E. coli cell contains about 5000 molecules of RNA polymerase. When the cells are growing rapidly, 2500 molecules will be bound to genes in transcription complexes. Another 1250 will be in initiation ...


1

Neither of the terms “Nucleotide-associated protein” or “RNA-polymerase associated protein” are standard in molecular biology in so far as they are not defined in the reference Gene Ontology. (In contrast, “DNA-binding” is.) An internet search does not bring up many examples of the former usage (at least) and in those it brings up it is not defined. One ...


1

I wouldn't call them hypotheses, but the question is intriguing, and, as it seems to be ignored in the literature, I'll make a couple of suggestions. Perhaps it has something to do with recognition of termination signals in relation to selective pressure for speed in the two processes. Perhaps if elongation of the RNA chain were any faster it wouldn't be ...


1

In this case you can run your PCR for another 18 cycles (if your total number of cycles was 30). However, do the initial denaturation for 1-2min. You may run the same program again (full 30 cycles) but you'll just end up wasting time because after a point all the dNTPs and primers would be exhausted and further cycles will have no effect on the concentration ...


1

Not sure if this question is still valid, but it was marked as unanswered and frequently viewed, so here's a go :) These days most polymerases (certainly for Real Time PCR) are so-called Hot Start enzymes (ask your manufacturer, because they save you a whole lot of trouble!). They are modified with a protein that prevents them from working until activated ...


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