I hope our laughter during the day was reassuring

C said that this site was getting too depressing...is it? =P
All the laughters had been spent in school that's why. HAHAHAH.

So I decided to write something happy here to balance it out.
Today is PTM day and we had such a good time talking behind the students' backs!! Heh! =)
But the greatest takeaway was knowing that the class has come along nicely and that we are now one big family if not working towards. And to know that even in my absence, they will be there for one another

MCQ answers only

I hope to be able to run through some of these questions in class.
But look through first k?!!!

thanks!

DNA & Genomics : Replication, Transcription, Translation

Disclaimer: most videos do not cover all the things highlighted in notes so just get a feel of the processes.


Replication
Video 1
http://www2.le.ac.uk/Members/jlb34/research/replication%207v3-3.swf/view
Video 2
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf::DNA%20Replication%20Fork
(however, this V2 lacks SS binding proteins!)

Question posed:
Why can't DNA polymerase (the other one) catalyse the formation of the phosphodiester bonds between the adjacent nucleotides of Okazaki fragments if it is able to elongate the Okazaki fragment after removing the RNA primer ? Why do we resort to DNA ligase?

Good Question!
We need to consider the nature of the 2: DNA polymerase and DNA ligase, both of which are enzymes.What is the important thing about enzymes? They have a substrate specific active site.
when DNA polymerase elongate the Okazaki fragments, the phosphodiester bond is formed between the free 3'OH end and a free nucleotide (nucleoside triphosphate). The reaction/formation of the bond also involve the removal of pyrophosphate (PPi) - the 2-phosphate group.

On the other hand, the substrate for DNA ligase is different. They are the free 3'OH end and a single 5' phosphate group extending from the adjacent fragment. The ligase only needs to join them up!

Thus although both involves the formation of phosphodiester bond, the different substrates involved demands the use of different enzymes.

Transcription

Video1:



Consider or note the following questions.
1) How are 2 DNA strands separated? (compare to transcription)
2) what happens to the 2 DNA strands as the RNA polymerase move along (and read) the template strand in the 3'-> 5' direction
3) what kind of nucleotides are involved? (compare to transcription)
4) where does transcription start? (compare to transcription)

Translation
Video 1:
http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter3/animation__how_translation_works.html
Video 2:
http://www.vcell.ndsu.edu/animations/translation/movie-flash.htm


Consider or note the following questions in understanding the process.
1) the mRNA is being read in what direction? (compare to R and T above)
2) In what sequence does the following binds to mRNA: small ribosomal subunit, large ribosomal subunit, initiator aminoacyl-tRNA carrying methonine.
3) Which site will methionine reside in initially?
4) By how much does the ribosome move each time?
5) What does the stop codon encode for? Or does it encode for anything?

Out of Syllabus (OFS) Questions

nO.1
R/S between tRNA, mRNA and rRNA in translation.
we had discussed extensively on the roles of tRNA and mRNA in translation but often skip rRNA. We only know rRNA as a component of ribosomes but what are they doing in ribosomes?

Let's appreciate the fact that different rRNAs are found in the 2 ribosomal subunits.


So what are their roles?
1) Peptidyl transferase is an enzyme found in the large ribosomal subunit that catalyse the formation of a peptide bond between the polypeptide chain (peptidyl-tRNA) in P site and the adjacent aa (aminoacyl-tRNA) in the A site of the ribosome. Now the elongated chain is now attached to the tRNA at the A site.
This peptidyl transferase is rRNA in action = it is a ribozyme, an RNA enzyme.

2) They are known to interact with
a) mRNA (rRNA in small subunit - translation step 1: small subunit binds to the mRNA..=)),
b) anti-codon regions and 3'CCA ends of the tRNAs at the P and A sites (rRNA in both subunits - no prize for guessing which for which).


3) also the subunit are able to interact because of interaction between the rRNAs in the 2 subunits!

COol yeah?!
Actually rRNA is not as well-studied so I will stop here.
_____________________________

nO.2
Proofreading mechanism of DNA polymerase in details

DNA polymerase has intrinsic 3'-> 5' proofreading exonuclease activity
(exo=outside; nuclease=enzyme that cleave nucleotides; exonuclease = enzyme that remove/cleave nucleotides from the terminal)

From Wiki:
When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3'-5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading).



How does the DNA polymerase know when there has been a mistake?

anticodon and codon pairing is very impt btw the tRNA (AC) and mRNA (C).
Incorrect base pairing --> conformation is altered/distortion in shape of the regular double helix which is recognised as a mismatch of base pair to initiate the exonuclease activity.


NOTE: not all DNA polymerase have proofreading capability (there are many types of DNA polymerase but the one we are interested in for replication has such capability)

________________________

Amendments and Otherwise

nO.1

Water Potential Notes
As pointed by D, on page 3, (c) pressure potential for plants only -
bullet number 3 - when a plant cell is in a solution with a higher (less negative) water potential, there is a net movement of water into the cell.

nO.2
Does nucleotide has one or three phosphate(s)?
It can be one, two or three, which also means that the term is non-specific.

To be specific, the raw material for DNA replication or transcription are deoxyribonucleoside triphosphates and ribonucleoside triphosphates.
Otherwise, we used the term free XXnucleotides very loosely.

You should know that nucleotide = nucleoside (pentose sugar + nitrogenous base) + phosphate grps

nO.3
Why are the dividing cells in tutorial so dark? Shouldn't there be only a dark spot known as nucleolus for cells in interphase?

Actually the diagram given wasn't so good. For cells in interphase, yes, you should be able to see the nucleolus quite well!:)

Cells in prophase are quite easily identified. I usually look for those in late prophase when you should be able to see individual chromosomes very well.
At interphase, you will not be able to see individual chromosomes.
One should note that mitosis is a continuous process so there are always intermediates stages.

PS: The chromosomes are usually stained so that one can see the them easily for the mitotic process.

http://www.vcbio.science.ru.nl/en/image-gallery/show/PL0096/labels/
http://www.microscopy-uk.org.uk/mag/artnov04macro/jronionroot.html
http://course1.winona.edu/sberg/IMAGES/anaphas.JPG
(for practice?)

Enzyme MCQs

LT test 2
D asked about using rate in length change to determine the rate of digestion. he pointed out that the change in length would decrease as the circle gets bigger.
I agreed. The method provided is a rather crude one (qn asked to estimate)
A better option is to use Area. In fact, we do have scripts that used area and we accepted it as well. =)

The Cat Is Out of the Bag

It finally jumped out, delivered in a mix-bag of sadness and relief.
Relief because it has been a weight on my mind for a while, unable to share and always trying to find the right moment. I wanted to let my R know during class camp but they were having such a good time that the moment never came. Then I realised perhaps there will never be a right moment.
Sadness because when it was out, it felt so real. I wasn't ready for it and wished I had more time instead of the short intermission to talk to R. But on hindsight, it was for the better before words get choked.
I am going to miss them more now after the class camp. Did they realise that they are a great bunch as one?

So the heart was a little squashed today.

Broke the news to all my classes. It is true. Saying farewell was not easy but at this age, I also know that it is not wise to leave your goodbye at the very end.
The only consolation is in knowing that even if I stay this year, I wouldn't be able to stay until the end of next year to witness their graduation.

Half-way through the day, I realised that being a teacher also means that even when you felt a little battered and wanted to sit down somewhere to contemplate, you still have to continue to ensure the smooth running of events for the students

---
I started the year tentatively,wary about getting too involved with the classes in case I do go off (after sending in my application in Jan) but it was difficult to keep that distance and not wanting to know them better. When the acceptance came as a surprise three wks ago, I wished that I could have done even more and better.
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Coming to the end of the week.
Like any other time, we have to make the best out of what ever we have.