BioMoo Seminar on Transcription: 7/3/96

JohnW turns the PPS-recorder on.

Jzt says, "Do the Cytosines, i.e. Montse, Karl, Horst, Dave have anything they want to discuss?"

Davidj says, "not yet thanks"

HorstJS says, "I have nothing special to discuss - I hope somebody else has ;-)"

KarlS [to jzt]: I do not have anything to discuss either JohnW is having trouble connecting to the Transcription material (gulp)

Raj [guest] says, "I understand transcription is todays subject"

JohnW . o O ( Ah, got it )

Ahotz says, "the promotor region of eukaryotic genes which is in preparation I missed"

Luis (idle 9m) has arrived.

HenryB [guest] [to ahotz]: yes this is the most complex part and is taking me time to put together

Raj [guest] says, "Phew !"

Montse [guest] "I think that Henry' s:I have the same problem with this part.

Jzt says, "Maybe Raj could do one of his lightening summaries of the main important points that we should take away from the course?"

JohnW says, "I would like to raise a point about sense and non-sense strands"

Ahotz says, "hat do you think about the "

Ahotz [to Luis]: yes

Raj [guest] says, "I'm not too up on transcription yet but my idea concerning the highlights are :"

JohnW says, "I.e., how is the sense strand always correctly identified during transcription, and does a continuous strand of DNA contain both sense and non-sense regions for different genes?"

Raj [guest] says, "1) Why transcribe in the first place ? Why the need for a secondary messenfger the first evidence for it - RNA polymerase enzyme of the year - How RNASe Pol functions with the DNA and RNA- direction of movement up the DNA, speed in bp per second- Realise that it has many functional domains including error cheking, etc- compare and contrast with DNA Pol - continuing ..."

HorstJS says, "to JohnW "I'm not sure about it but I think, that both strands are used for coding...'"

HenryB [guest] [to JohnW]: no DNA strand is either sense or antisense. Gene sequences can be found on both

HenryB [guest] [to ]:

Davidj says, "I believe that in some viruses not only both strands but opposite strands from the same region code for different proteins!"

HenryB [guest] says, "

Raj [guest] says, "3 types of RNA Pol I II and III, antibiotic inhibitors of transcription; How could RNA pol help us understand the evolution of replication from the RNA world...."

HenryB [guest] [to ]:

Ahotz [to davidj]: that is what I know, too

HenryB [guest] says, "one strand can act as boith sense and antisense"

Raj [guest] says, "The Central Dogma of Mol Biol - Reverse Transcriptase - every Dogma must have its day - Retroviruses"

HenryB [guest] says, "the sense strand is defined by the diredtion the promoter points in"

ClareS . o O ( that's what I thought, but I wasn't quite sure ) JohnW nods

HorstJS says, "(cont. Davidj) In some viruses (eg Phi X174) you have even overlapping genes on the SAME strand'"

JohnW says, "Ok, that makes sense ('scuse pun)"

Raj [guest] says, "Possibly approaches to AIDS therapy using HIV rev trans. inhibitors etc... zzzzzz"

HenryB [guest] says, "in SV40 you find that all three sframes are used at once (but only for a very shirt distance)"

Raj [guest] says, "I think thats it for the time being"

JohnW [to HorstJS]: Ah yes, this is the virus where all possible reading frames are used?

HenryB [guest] says, "yes but only for a very short distance. viruses need to make the most of their limited coding ptential"

CrisCan says, "I have a problem with the regulation of prokaryotic genes. may you confirm that the negative regulation of lac operon is working togheter with the positive regulation of the operon by AMPc?"

HorstJS says, "to JohnW "I don't know exactly - I'ts about 10 years ago, that I heard that Story of Phi X174'"

Davidj says, "there has been quite an exhaustive discussion of the terms sense/antisense/coding/non coding on the Bionet newsgroups recently, followed by a vote for what to adopt as standard nomenclature. I can't remember the details but it will be summarised, probably, in Paul Hengen's writings in TIBS"

Ahotz says, "@more"

ClareS [to davidj]: Which Bionet newsgroups?

HenryB [guest] [to CrisCan]: yes in the presence of lactose err this is a mess I will try again

Davidj says, "it was methods and reagents, I believe"

JohnW says, "This overlapping reading frame business raises interesting questions regarding selection"

HenryB [guest] [to CrisCan]: Inthe presence of lactose the repressor no lonfger binds to the DNA but transcription is dependent on the absence of glucose ie presence of CRP+cAMP

Raj [guest] says, "I would just like to throw in a bit of philosophy- The lac Operon is interesting because it seems to reflect a semblance of decision making on behalf of the whole organism which is clearly understandable at the reductionist level- thats why Jacques Monad got all excited and wrote the depressing "chance and necessity" (i.e. "(wo)man finds her (him)self alone in a cold lonely cosmos governed only by the laws of chance and necessity")"

HorstJS says, "to JohnW "What questions for example?"

CrisCan [to HenryB]: ok but what does it happen when both lactose and glucose are absent and there is an other sugar?

HenryB [guest] [to JohnW]: it does, doen'y it. The requirement to maintain threee functional protiens from one coding sequence

HenryB [guest] [to CrisCan]: sorry your last message was corrupted at the important point. E coli uses glucose in preference to anything else

HenryB [guest] [to ]: CerisCan

HenryB [guest] [to CrisCan]: if there are tow alternative sugars available eg lactose and galactose I gues it would try and use both The housekeeper arrives to cart tday off to bed.

JohnW is digesting Raj's philosophy

CrisCan [to HenryB]: if there isn't either glucose (CPR is able to bind DNA) and lactose is absent (the repressor block lac operator) lac couldn't be transcript. is this true?

Raj [guest] says, "its not my philosophy, I just thought it interesting"

Raj [guest] says, "Do you all know that mRNA was last to be discovered because it has a much higher turnover rate (which is used to regulate protein synthesis) than ribosomal and transfer RNA"

Raj [guest] says, "also it is very hard to work with RNA in the lab than with DNA" KarlS has disconnected.

HenryB [guest] [to Raj]: is that comment made from bitter experience? ClareS is regretting not having studied biochemistry to degree level! Ahotz says, "yes, RNA is degraded very easily"

Raj [guest] says, "no , Ive only worked with DNA but I have heard some horror stories" Ahotz says, "but it has to have a high turnover that the cell can change the protein set" HenryB [guest] says, "take my word for it - they are all true" Davidj says, "does mRNA have special motifs that mark it out for rapid turn over comparable to PEST sequences in certain proteins?" HenryB [guest] says, "yes usually buried in the 3' untranslated sequence. Som signify rapid turnover "

Raj [guest] says, "I would like to kow why RNA is degraded more than DNA ; what is the structural reason, surely the lack of one oxygen atom on the sugar of DNA compared to RNA doesnt explain this or does it or is it because we secrete RNAses as do other organisms and these get into the preps. Has anyone worked with RNA ?"

Raj [guest] says, "what is a pest sequence" Davidj says, "Thanks HenryB, the next question is, is it that RNA is more sensitive than DNA or RNAses more resistant than DNases?" Ahotz says, "I have isolated mRNA a few times" HenryB [guest] says, "yes all the time. The most prevalenyt problem is DxxRNAse" HenryB [guest] says, "we secrete it in sweat which is why RNA workers wear gloves alll the time" Davidj [to Raj]: Pest sequences are rich in certain aminoacids including Proline and serine? that mark a protein out for specific ubiquitin-mediated proteolysis (I think :-))

HenryB [guest] says, "DNAses equire Mg RNases don't. Also RNAses are difficult to sedstroy even by boiling"

Raj [guest] says, "{to David} thanks, I'm still learning protein degradation"

Raj [guest] says, "I know we secrete lysozyme in our tears, does it also contain RNAses"

DavidJ says, "you prepare DNase-free RNase in the lab by boiling it to destroy any DNase activity"

Raj [guest] says, "Its all coming back now"

HenryB [guest] says, "My final point about RNA and DNA stability is that the 2 adjacent OH groups in RNA make itmore labile to OH hydrooysisi"

Ahotz says, "you should autoclave to destroy RNase"

HorstJS says, "to Raj"As far as I learned it - it is more or less only the 2 prime OH-Group in RNA which makes it so hydrolysis'"

Raj [guest] says, "Anyone know the difference between RNAse A and T ?"

ClareS says, "I discovered that one of the sugar transporters I was working on was quite similar to one particular PEST containing sequence, but I had *no idea* of the function of these proteins before. Thanks!"

Raj [guest] says, "[to JSH thanks for that insight]" The housekeeper arrives to cart KarlS off to bed.

HorstJS says, "Just deprotonate the 2prime OH by NaOH and you can do a perfect intramolecular Sn-Hydrolysis on the Phosphate - which is very fast because it's Intramolecular"

HenryB [guest] [to Davidjlost]: what I started to say - forget oit

Davidj [to Raj]: whats RNase T, my crib book only lists A and H?

Raj [guest] says, "[to David J] I used RNASe T1 a few years back along with RNAse A. A comes from sigma (calf thymus) and T (also in Sigma catalogue) I think may be recombinant based on a T phage protein (speculating wildly here) but the point is that one cuts RNA into large length pieces and the other into smaller ones- different recognition sequences- perhaps a more common 4-mer for RNAse A and a less common 6-mer for RNASe T"

HenryB [guest] says, "RNAse A and T differ in sequence specificty. RNAse H degrades the RNA strand in a RNA/DNA hybrid"

Raj [guest] says, "If we are drying up can we talk about reverse transcriptase ?" JohnW nods

HenryB [guest] says, "whty not"

Raj [guest] says, "I guess when it was found that the central dogma was violated a lot of lamarckists must have rejoiced because it hinted that environment could feed back into nucleic acid"

JohnW says, "could you elaborate?"

HenryB [guest] says, "and you can find sequence s in human DNA which are obviously derived from a mRNA"

JohnW . o O ( ok, I see )

HenryB [guest] says, "but despite the fact that these obviously were mRNA at one time they are no good as genes because thy lack a promoter"

Raj [guest] says, "I mean: if the only way diversity comes into the world is by the selection of random DNA mutations being selected and made into proein, R.T makes the jump back from RNA to DNA (but not from protein to RNA which is what lamarckists would like - for example- so that ironsmiths would pass their strong arms to their kids- and make Stalin very happy"

JohnW is digesting that

Ahotz has disconnected.

HenryB [guest] says, "My life as a molecular bilologist would be made far easier if somebody did find a reverse translatease rto turn protin into RNA@!!"

Raj [guest] says, "Jzt is clear to land on runway no. 2, wind is 5 knots easterly, surface conditions wet"

JohnW . o O ( ??? )

Jzt [to Raj]: jt is not jzt - I'm still sweating it out here

DavidJ says, "maybe the "dogma" was a bit overenthusiastic in going DNA to RNA to Protein, maybe it should have been more conservative ie Nucleic acid to protein. I think that the DNA is a bit like a very precious treasure map, you wouldn't take it with you on a treasurwe hunt in case it got lost/damaged/destroyed, you make a copy and take that instead, so you can always go back and make another instead (taken from the DAJ noddy guide to DNA:-))"

JohnW smiles

HenryB [guest] says, "if you believe the evolutionary biologists then all life started with RNA. Then we evolved DNA to act as genetic material and proten as enzymes"

Raj [guest] says, "DNA is often referred to as a blueprint (sort of map), P W Atkins in the " Creation" draws attention to the difference between a blueprint and a recipie. Is a recipie a better metaphor or similie etc ?" Montse [guest] has disconnected.

The housekeeper arrives to remove Montse [guest].

DavidJ says, "so which came first DNA, RNA or, heaven forbid protein? Whilst we can't comprehend the antidogma, could we swallow nucleic acids evolving as a way of making existing peptides formed through non "living" processes?"

HenryB [guest] says, "viruses have RNA as genetic material. Robozymes )RNA enzymes) exist and these can replicate RNA so RNA was first" The housekeeper arrives to cart ahotz off to bed.

HenryB [guest] says, "fredian mistake robozymes instaed of ribozymes"

DavidJ says, "is a recipe or more like origami instructions where simple instructions can make comlicated shapes and bring previously distant regions together?"

JohnW says, "think sounds like a superhero :)"

Raj [guest] says, "I cant get my head round chicken and egg conundrums, its tempting to think that it would take a lot longer than 4.5 billion years and maybe it all started somewhere else in outer space. I know this doesnt help scientifically but it all seems so clever and unaccountable to me"

Raj [guest] says, "I don't believe in Freudian slip it ins (oops)"

Davidj [to Raj]: maybe somewhere out in the cosmos, one of the infinite number of Raj's has just seen the light:-)

Raj [guest] says, "I like the recipie idea because it includes a temporal dimension. Also a small change in ingrediants can drastically change a cake"

HenryB [guest] says, "is that not also true of a blueprint?"

Raj [guest] thinks ..

JohnW says, "I suppose the concept of a blueprint is a representation of a end-product, which could be produced in any one of many unspecified ways."

DavidJ says, "How much of a temporal dimension do we need? How fast does translation occur?"

Raj [guest] says, "no, a small change to a building plan will be a small change in the finished structure"

HenryB [guest] says, "if you misread the sacle and think cm = m then this is a big change"

HenryB [guest] [to Davifdj]: don't know how fast translation is but I would guess one or twop minutes

DavidJ says, "if we are talking metaphors, what about an impressionist painting, made up of discreate units where it is the order (position)in which they are assembled that gives the final product"

Raj [guest] says, "in size but not in form, the extra information needed to describe the larger structure would be a tiny scaling factor (a few bits of code) but the difference between a meringue and a cookie is greater in information terms- i.e. greate complexity from a small change"

Raj [guest] says, "Any one want to discuss SNURPs ?"

DavidJ says, "its funny, but whenever we hold these sessions to discuss proteins, we end up talking DNA"

JohnW laughs

HorstJS grins.

Raj [guest] says, "we shouldn't have read so much Richard Dawkins"

HenryB [guest] says, "Don't n be selfish , gene !!!" CrisCan has disconnected.

ClareS says, "I don't know what a SNURP is, I'm afraid!"

Raj [guest] says, "I think most people have a gut feeling for thinking proteins are a way of DNA to make more DNA. How would it feel to actually think of DNA as a proteins way of making more protein ?"

Raj [guest] says, "small nuclear ribosomal particl"

ClareS nods

Raj [guest] says, "they are made of RNA and were exciting to find because they process the introns out of mRNA sequences in eukaryotes. Up till then only proteins were believed to have enzymatic (catalytic) properties"

ClareS says, "so snurps = ribozymes, or am I missing something obvious?"

Raj [guest] says, "I guess ribozyme is a generic term (rna enzyme ) so Snurps are atype of ribozyme" TaekKwon [guest] finds its way in.

HorstJS says, "do Snurps not contain any protein??"

Raj [guest] says, "{to clar and others interested} Scientific American have published some good articles on snurps in the last 5 years "

HenryB [guest] says, "yes snurpos are ribonucleoproteins. Theydo not havwe ribozyme activitity"

Raj [guest] says, "but ribosomes had been known for years to contain RNA and protein so why the fuss on the discovery of snurps ?"

DavidJ says, "do ribozyme genes contain introns? Logically they shouldn't"

Raj [guest] says, "could it be that the RNA in a ribosome is purely structural whereas in a snurp it is involved in the processing reaction (which i thought would qualify it as catalytic RNA)"

DavidJ says, "and what prevents ribozyme RNA from being translated?"

Raj [guest] says, "presumably it must lack the translation initiating codon"

Jzt has disconnected.

Raj [guest] . o O ( I must be getting the bus home soon )

JohnW . o O ( yes, this has been a meeting and a half )

Raj [guest] waves goodbye and disapearing on the horizon, as the hour is late he casts a long shadow

HenryB [guest] says, "mRNA that has to be tgrasnlated has a seriwes of signals that tell the ribosome what to do u! RNA in sunros does not hzave these signals"

HenryB [guest] says, "I relallt"

HenryB [guest] says, "I really must learn to tupe"

Raj [guest] has disconnected.

HenryB [guest] says, "U1 RNA has no signals in it for translation"

Davidj [to HenryB]: thanks

JohnW says, "I will also have to go soon."

HenryB [guest] says, "me too. they lock my building soon"

HorstJS says, "me too"

ClareS says, "Me too..."

DavidJ says, "me too, the wife is already threatening to change the locks, oh the joys of long distance commuting :-)"

DavidJ says, "Bye all, and thanks"

Davidj has disconnected.

JohnW says, "Ok, we shall bring this to a close. I would like to thank Henry for attending. Henry, I hope you now have some insight into the joys of BioMOO, even if it is rather fiddly at first !"

JohnW smiles

HenryB [guest] says, "godbye I mean goodbye all"

HenryB [guest] [to JohnW]: yes but I need to imporve my typing. Thanks fror thwe experience. Bye

ClareS says, "thanks, Henry!"

JohnW says, "Thanks to everyone here too, for sharing your expertise. I will leave the recording of this meeting here."

HorstJS says, "Thank you very much henry"

HenryB [guest] [to JohnW]: How do I disconnect?

JohnW says, "if you type '"

HenryB [guest] says, "Btye all. I enjoyed it. See you agaunfg again soon"

JohnW says, "@quit"

HenryB [guest] says, "@quit"

ClareS [to HenryB]: without the "s !!

HorstJS says, "Goodbye to everyone - till the next meeting!! :-)"

JohnW says, "then you will exit BioMOO."

HenryB [guest] has disconnected.

HorstJS slowly gets translucent and vanishes in a small cloud of fog

The housekeeper arrives to remove HenryB [guest].

HorstJS has disconnected.

JohnW says, "Bye, and heres to the next discussion meeting. Any preferences for topics, let us know on the e-mail lists..."

JohnW says, "look"

JohnW . o O ( oops )

JohnW turns the PPS-recorder off.

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