During transcription in eukaryotes…

…the poly(A) sequence serves to signal to auxiliary enzymes to snip off the nascent mRNA and add the poly-A tail. 

But the polymerase itself keeps going, synthesizing a “junk” RNA strand. How is the activity of this polymerase terminated?

There are two models: in the torpedo model, exonuclease runs along the growing excess RNA strand in a 5’ to 3’ direction, degrades it, and eventually knocks the polymerase off of the DNA template. In the allosteric model, transcription of the poly(A) signal induces a change in the RNAP complex (potentially by recruiting a destabilizing protein, for example).

These models seek to explain an interesting process in eukaryotic transcription, and the exact mechanism likely lies in a hybrid model. Arguably even more interesting than how the process occurs…is why? Why has an “overtranscribing” approach been adopted for eukaryotic transcription?

The authors of the aforementioned review suggest this: “This unique and complex mode of termination by Pol II may have arisen to allow for transcription of very long genes without prematurely stopping at terminator-like sequences that occur by chance throughout the gene.”

Cold and menthol sensitive receptor (CMR1)

How exactly do we sense cold? On the molecular level, if cold is just the absence of heat - what enables our cells to respond? 

It turns out the answer to this question is also the answer to a different one: why does menthol (found in cough drops) elicit a soothing reaction when consumed/applied to skin?

Surprisingly, both cold and menthol act on the same receptor: a 1,104 amino acid long protein ion channel found in the membrane of certain receptor cells, known as CMR1. Presumably, the binding of menthol and a decrease in temperature cause conformational changes in CMR1, allowing a rapid transfer of ions (such as calcium) and leading to the feeling of cold. In 2002, researchers published a paper in Nature describing how they identified and characterized this protein.

Further work done in 2007 provides support that CMR1 (aka TRPM8) is the main detector of cold. 

It’s important to note, however, that CMR1 is primarily responsible for the detection of temperatures 8-28C (or 46-82F). For freezing temperatures, other receptors are likely involved. When the CMR1 in mice was disrupted, the mice still retained some ability to avoid extremely cold surfaces, though temperature detection on the whole was severely impaired. 

A basic diagram of the protein and methanol is shown below. Interestingly, the CMR1 belongs to a larger family of related ion channels, one of which mediates the binding of capsaicin (what makes things spicy!).

The nature of DNA Polymerase (cont.)

In our first post about DNA polymerase, we went over the reasoning behind the lack of a 3’ to 5’ polymerase. The goal of this second post is to understand another peculiar quality of DNA polymerase - the absolute need for an RNA primer. 

Replication cannot begin without such a primer; there is no known DNA polymerase that can begin elongation from scratch. DNA replication throughout all domains of life requires an enzyme (DNA primase - shown below) to first synthesize a short RNA primer.

Is a DNA Polymerase enzyme that does not require a primer simply too complex for nature to produce? Probably not - consider DNA polymerase’s cousin, RNA polymerase, which can initiate synthesis of an mRNA strand de novo. So it is likely that such a DNA polymerase existed - however, as it has not been found in any organism yet, we can conclude that the cons of having such an enzyme far outweighed the pros.

Comparing RNA and DNA polymerase once more, we note that RNA polymerase has relatively limited proofreading abilities. This makes sense when we consider the millions of RNA strands that are transcribed - one faulty strand won’t make a big difference. In comparison, DNA polymerase must have exceptional proofreading abilities (1 x10^9 errors per nucleotide polymerized); errors in DNA are usually highly detrimental. We can argue, then, that the ability to synthesize strands de novo and an enzyme’s proofreading ability are linked.

An enzyme (such as RNA polymerase) capable of synthesizing strands from scratch gives up some copying fidelity, while an enzyme (such as DNA polymerase) capable of proofreading gives up the ability to start a new strand. 

Using this argument, we can also figure out why the primers for replication are made of RNA, instead of DNA. DNA primase shares characteristics similar to RNA polymerase - it has the ability to start synthesis anew; thus, it will likely have a higher error rate. If DNA primase used a primer comprising DNA nucleotides, the error-filled primer would then be incorporated into the genome.

To preserve genome integrity, a DNA primase that specifically uses RNA nucleotides has been selected for, as RNA is not incorporated into the genome. Thus, this RNA-using DNA primase can synthesize the necessary primer for the more accurate DNA polymerase to work off of, while at the same time ensuring its faulty primer is not incorporated into the genome. In later steps, the RNA primer is removed and replaced by DNA.

Source:

Molecular Biology of the Cell, Fourth Edition. (2002)

The nature of DNA Polymerase

This incredible enzyme replicates the immense (billions of bases long) DNA library found in every one of your cells, making perhaps only one error per billion nucleotides polymerized.

Two unique characteristics set this enzyme apart from other proteins: the need for an RNA primer, and its exclusive 5’ to 3’ activity. Though these two characteristics may seem puzzling, there is actually a clever molecular logic behind them.

Part of what makes DNA polymerase so accurate is its inherent “proofreading” ability. When the enzyme adds an incorrectly paired nucleotide to the strand, it can detect and remove the error before continuing elongation. Herein lies an explanation for the lack of a 3’ to 5’ polymerase.

The energy for replication is supplied by free-floating nucleotide triphosphates - specifically, the hydrolysis of the pyrophosphate group supplies the energy for one more base to be added.

In 5’ to 3’ replication, the 5’ triphosphate of a free nucleotide reacts with the 3’ hydroxyl group of the elongating strand (as depicted above on the left). A phosphodiester bond is formed between the two nucleotides. If a mistake is made, the polymerase removes the incorrect nucleotide and adds the correct one. The key point here is that the 3’ hydroxyl group of the elongating strand is always open to react with the next nucleotide; the energy for the reaction comes from the 5’ triphosphates located on the free-floating nucleotide triphosphates.

This would not be the case with a hypothetical 3’ to 5’ polymerase. Elongation would involve a reaction between the 5’ triphosphate of the elongating strand and the 3’ hydroxyl of a free nucleotide. In this scenario, the energy for the reaction comes from the triphosphate located on the strand. 

(3’ End of strand—-Nucleotide—-5’ Triphosphate) + (3’ OH—-Nucleotide—-5’ Triphosphate)

—-Elongation reaction—->

(3’ End of strand—-Nucleotide—-Phosphodiester bond—-Nucleotide—-5’ Triphosphate) + (pyrophosphate)

If a mistake were made, the mismatched nucleotide would be excised - but there would no longer be a 5’ Triphosphate end.

(3’ End of strand—-Nucleotide—-Phosphodiester bond—-Mismatched nucleotide—-5’ Triphosphate)

—-Exonuclease reaction—->

(3’ End of strand—-Nucleotide—-5’ Phosphate) + (Mismatched nucleotide—5’ Triphosphate)

After the removal of the mismatched nucleotide, all that would be left is a single phosphate on the 5’ end of the new strand, which cannot power the reaction.

As a result, the 5’ to 3’ polymerase has been selected for because it maintains DNA copying fidelity. The 5’ to 3’ nature of polymerase combined with the antiparallel nature of DNA has led to the well-known lagging and leading strand phenomena (which has inadvertently given biology students many headaches!) 

Thymine vs. Uracil

We all know from high school bio that thymine takes the place of uracil in DNA. But why exactly does this occur? What advantage does thymine offer over uracil?

When I asked a teacher about this, she replied: Thymine is a more stable molecule than uracil, and thus helps maintain the integrity of DNA. 

However, the only difference between these two bases is a single methyl group:

So how exactly does that small change stabilize DNA?

For one, it prevents DNA from being recognized and chopped up by nucleases - the methyl group thus helps to protect DNA against invaders. The hydrophobic effect of the methyl group also helps to ensure proper base pairing (uracil can occasionally pair with other bases). Thymine’s methyl group also provides a point of interaction for amino acids in proteins (possibly resulting in better recognition by polymerases, transcription factors, etc).

Perhaps the strongest impetus, though, for the incorporation of thymine into DNA comes from the spontaneous deamination of cytosine. This undesirable chemical reaction results in a uracil base, and occurs, on average, 100 times per day in a mammalian cell.

The buildup of these “illegitimate” uracils could be catastrophic for the organism - at the very least, copying fidelity of DNA would be detrimentally affected. Thus, cells have repair systems in place to remove these “illegitimate” uracils. But if uracil were already present in DNA, paired to adenine, the repair system would be forced to somehow differentiate between “illegitimate” and “legitimate” uracils. An easy solution to this problem? Add a methyl group to all of the “legitimate” uracils, allowing the repair system to easily tell between the two. This usage of methylated uracil, or thymine, in DNA allowed for the long-term storage of crucial genetic information. 

Sources / Further reading:

Uracil vs. Thymine - Molecular Station forum

Why does uracil replace thymine in RNA? - MadSci

Ivarie, R. Thymine methyls and DNA-protein interactions.

Lehninger - Principles of Biochemistry, Third Edition (2000)

So what is molecular biology, anyways?

Molecular biology is often labelled as the branch of life science which considers biological activities and functions on the molecular level. So using xkcd’s little comic down there as a guide, we can put molecular biology somewhere in between the biologist and the chemist (the best of both worlds?). Why has this field blossomed so rapidly? Observable complex macroscopic phenomena (such as homeostasis and consciousness) stem from the plethora of interactions that occur at this molecular level.

Unsurprisingly, to study the hubbub of molecules bouncing around in the cell, molecular biologists must utilize a large palette of skills and techniques. Take Francis Crick for example - he began calling himself a molecular biologist after he “got tired of explaining that [he] was a mixture of crystallographer, biophysicist, biochemist, and geneticist”! 

The allure of molecular biology has been such that more and more scientists are now drawn to the ongoings of the microscopic biological world. Molecular biology also offers great potential for the treatment of diseases and overlaps with other “hot” fields, such as genetics and biochemistry. Fruitful collaborations with other scientists (bioinformaticians, chemists, and even physicists) are becoming increasingly more common. And perhaps most importantly, advances in this field have led to paradigm shifts in the way we view inheritance and gene expression.

Some notable achievements:

- RNA Interference: Nobel lectures by Andrew Fire and Craig Mello

- The reprogramming of mouse skin fibroblasts into induced pluripotent stem cells from just four transcription factors

- Advances in the burgeoning field of epigenetics

…and a few of the big questions and challenges that still remain:

- What is the purpose of “junk,” or noncoding, DNA?

- Connecting findings from the cellular level to the pathology of diseases on the organismal level

- Transcriptional regulation: how do methylation, miRNAs, etc. interact to control gene expression?

This is only a woefully brief list of the various topics of interest in molecular biology today. It’s likely that research being conducted this very second will lead to the birth of new fields! Whatever may be the case, molecular biologists will doubtless be the vanguard leading us into the century of biology.

Sources / Further reading:

Contributions of Molecular Biology to Medicine by Francis O. Schmitt

A decade of molecular cell biology: achievements and challenges by Akhtar et. al

Scientific convergence in the birth of molecular biology by Sunny Y. Auyang

Wikipedia has a nicely written article on the molecular biology as well.

What are the top 125 questions that remain unanswered in science?