DNA replication, Self splicing RNA...

[sps27]

1) In Self Splicing RNA method which is basically removing the Introns from the newly transcribed 'Nascent RNA', I read that tRNA was used. A molecule such as Guanosine-OH recognizes the A-G or a U-G sequence. Does that identify an Intron? How are Introns identified in 'Nascent RNA'? Also, once the Introns are spliced out, what happens to them? Do they degenerate in the nucleus? Why is G-OH molecule considered an RNA?

2) During DNA replication, the first dNTP is added to the 3'-OH end of the primer. And then subsequently more keep getting added to the 3' end of the new fragment by DNA Polymerase 3. So does that mean the primer end will be towards the 5' end of the new DNA and the new DNA is essentially keeps growing the 3' end, which is why they say the new DNA is made 5' to 3'. Is this understanding correct?

3) Is the role of DNA Polymerase 1 which is splicing the primers and adding 20 dNTD's(deoxy-nucleotides) in place only limited for 'lagging' strand? Does it do the same with the 'leading' strand as well? The leading strand does not need any joining of fragments so I take it that DNA Ligase does not act on the leading strand. Right?

4) Are these Introns likely to be found more in the G-C heavy region of the RNA as opposed to U-A?

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[ChodeNode]

1) In Self Splicing RNA method which is basically removing the Introns from the newly transcribed 'Nascent RNA', I read that tRNA was used. A molecule such as Guanosine-OH recognizes the A-G or a U-G sequence. Does that identify an Intron? How are Introns identified in 'Nascent RNA'? Also, once the Introns are spliced out, what happens to them? Do they degenerate in the nucleus? Why is G-OH molecule considered an RNA?

2) During DNA replication, the first dNTP is added to the 3'-OH end of the primer. And then subsequently more keep getting added to the 3' end of the new fragment by DNA Polymerase 3. So does that mean the primer end will be towards the 5' end of the new DNA and the new DNA is essentially keeps growing the 3' end, which is why they say the new DNA is made 5' to 3'. Is this understanding correct?

3) Is the role of DNA Polymerase 1 which is splicing the primers and adding 20 dNTD's(deoxy-nucleotides) in place only limited for 'lagging' strand? Does it do the same with the 'leading' strand as well? The leading strand does not need any joining of fragments so I take it that DNA Ligase does not act on the leading strand. Right?

4) Are these Introns likely to be found more in the G-C heavy region of the RNA as opposed to U-A?

1 and 4, I don't know.

2) Are you asking the reason it goes that direction?. My understanding is that the DNA Polymerase-1 is reading the template strand from 3' to 5'. Because DNA is anti-parallel, as you read 3' to 5' on the template strand you are moving from 5' to 3' on the strand being built. The fact that it is anti-parallel is why it writes in the opposite direction it reads.

3) That I know of, there's no difference in function for DNA Polymerase 1 based on strand. The lagging strand needs to get "sewn up" by DNA Ligase because of the Okazaki fragments that are created. Look at some animations on this process and it will help solidify why Okazaki fragments occur. Or better yet, get good enough to draw it out for yourself.

 

[sps27]

1 and 4, I don't know.

2) Are you asking the reason it goes that direction?. My understanding is that the DNA Polymerase-1 is reading the template strand from 3' to 5'. Because DNA is anti-parallel, as you read 3' to 5' on the template strand you are moving from 5' to 3' on the strand being built. The fact that it is anti-parallel is why it writes in the opposite direction it reads.

3) That I know of, there's no difference in function for DNA Polymerase 1 based on strand. The lagging strand needs to get "sewn up" by DNA Ligase because of the Okazaki fragments that are created. Look at some animations on this process and it will help solidify why Okazaki fragments occur. Or better yet, get good enough to draw it out for yourself.

Thanks! I should have said that these facts I have mentioned are from TBR Bio book, chap Expression of Genetic Information.

regarding 2) I was trying to understand when the leading strand is duplicated, which end of the duplicated DNA are the new nucleotides attached to i.e., 5' v/s 3'. It seems to me they are attached to the 3' end. The 5' end of the new DNA strand keeps getting further and further away from the replication fork. And so the primer for that strand is closer to the 5' end. Just wanted to confirm this understanding, that's all....

regarding 3) I thought that DNA Polymerase 1 would not be necessary for leading strand as it is not fragmented. But it still has a primer which needs to be removed, so maybe it is reqd for the leading strand as well. Wanted to confirm that as well....

 

[mcloaf]

There are several different splicing methods that you seem to be mixing together a bit.

Most introns are spliced out by spliceosomes, which are RNA-protein complexes. Some rare introns have intrinsic splicing activity, which is the mechanism involving G-OH (group I self splicing introns) you mentioned. There is also another rare splicing mechanism for tRNAs. The wikipedia page on RNA splicing gives a nice explanation of the different mechanisms.

Basically, there are internal splice sites called splice donors and splice acceptors that are recognized by the spliceosome/intrinsic splicing activity/etc. The RNA is cut at the splice donor (5') and splice acceptor (3') and ligated together, and the intervening sequence is the intron. These introns are then usually just degraded, though they can also be processed into mirtrons (but that's beyond the MCAT for sure). What's cool is that mutations to these splice sites can actually cause disease.

#2 is easy: nucleotides are ALWAYS added 5' to 3'.

#3: You're correct in that the primer will need to be removed, replaced with DNA, and ligated on the leading strand as well as the lagging strand. The only difference is that there are many sites where this needs to happen on the lagging strand, and only one site per replication fork on the leading strand.

#4: I don't know, but I can't imagine why they'd be favored in a GC rich region. Did you read something that made you think they would be?

 

[sps27]

There are several different splicing methods that you seem to be mixing together a bit.

Most introns are spliced out by spliceosomes, which are RNA-protein complexes. Some rare introns have intrinsic splicing activity, which is the mechanism involving G-OH (group I self splicing introns) you mentioned. There is also another rare splicing mechanism for tRNAs. The wikipedia page on RNA splicing gives a nice explanation of the different mechanisms.

Basically, there are internal splice sites called splice donors and splice acceptors that are recognized by the spliceosome/intrinsic splicing activity/etc. The RNA is cut at the splice donor (5') and splice acceptor (3') and ligated together, and the intervening sequence is the intron. These introns are then usually just degraded, though they can also be processed into mirtrons (but that's beyond the MCAT for sure). What's cool is that mutations to these splice sites can actually cause disease.

#2 is easy: nucleotides are ALWAYS added 5' to 3'.

#3: You're correct in that the primer will need to be removed, replaced with DNA, and ligated on the leading strand as well as the lagging strand. The only difference is that there are many sites where this needs to happen on the lagging strand, and only one site per replication fork on the leading strand.

#4: I don't know, but I can't imagine why they'd be favored in a GC rich region. Did you read something that made you think they would be?

Thanks for your explanations. I will read the Wikipedia page on slicing. Yes, I am a little muddy on those concepts. Let me get back to you on 4). I thought I read somewhere in TBR on those lines but let me confirm that first. Thanks once again.

 

[sps27]

#4: I don't know, but I can't imagine why they'd be favored in a GC rich region. Did you read something that made you think they would be?

I was wrong about this. I muddled up Introns with termination sequence found on the DNA template - which is a G-C rich region followed by a A-T rich region. The RNA Polymerase recognizes that as a termination sequence.