weber uiuc edu 15 October 2007 MCB150 Lecture 22 Lecture 22 15 October 2007 Announcements More broken hand notes Exam II Thursday 5PM Check online for room assignment Regular lecture on Monday Monday night at 5 7PM in 228 NHB Review Review on Wednesday too o No new lecture content on Wednesday Office hours o Tuesday 12 2 o Wednesday 3 5 o May be in other location check in his office first WebCrossing deadline to answer questions 10 00pm Ribosomal Genes in Tandem Arrays Context of functional domains of the nucleus o Falls under central dogma o Information on how things are done within the cell The nucleolus is a functional domain highly organized It exists because regions of chromosomes especially in humans have come together then are surrounded by proteins etc to bring together regions of tandem repeats o It is not the entire chromosome just the segments containing genes for the 5 8S 18S and 28S rRNA o Nucleolar Organizing Regions NOR The nucleolus looks like it disappears during M phase o This is because the chromosomes are pulled apart to replicate o If rRNA synthesis is not occurring then the nucleolus does not exist We can have as many as 10M ribosomes per cell o Need to make lots of these o No amplification is possible Remember ONLY mRNAs are translated rRNAs and tRNAs are not translated Only transcribed o Therefore the cell needs to do transcription rapidly and efficiently MICROGRAPH o See examples of rRNA being made in parallel in the TEM micrograph in the notes It looks like a string of Christmas trees Promoter region is at the top RNA poly I will start on promoter using specific transcription factors to get it started and moving As soon as it is far enough away the next round of TFs start up and so forth o The branches are transcripts the rRNA that is forming being transcribed o The branches are longer at the end because they have been worked on longer Page 1 of 6 weber uiuc edu 15 October 2007 MCB150 Lecture 22 o Remember these are back to back copies of identical transcribed information So the next set is started immediately We only see three here but there are many many more o The beads on the end are not ribosomes translating These are not mRNA They are rRNA Instead these are the processing of the subunits Molecules jumping on and starting their work in organizing the structure of the RNA o This is NOT considered polysistronic because we are not making protein o Proteins are associated with the rRNA here in the nucleus to make the ribosomes Tandem Arrays We will end up with tandem arrays producing 28S and 18S and 5 8S o These are transcribed in one shot o We will end up with three pieces but we start by making one giant piece o That is one 45S piece is cleaved to make the 28S 18S and 5 8S subunits This is called a pre rRNA o The primary transcript of a NOR is a gigantic piece of rRNA that is 45S o We don t use the 45S pre rRNA directly We will process the molecule o We do not splice the 45S pre rRNA we simply cleave it rRNAs ARE NOT SPLICED THEY ARE CLEAVED It is cleaved into 5 8S 18S 28S molecules o We do not need to know the steps but understand the steps that are being done o In a step wise fashion we do an endonucleaic cut ETS external transcribed spacer Will not end up in an RNA molecule ITS internal transcribed spacer These are part of the primary transcript but not used o There is no splicing because the chunks in gold are not attached to one another KNOW WHAT WE START WITH AND WHAT WE END WITH 45S 5 8S 18S 28S What was just described here may make more sense if you read the next slide Revisit Previous Slide The genes for the 45S pre rRNAs are found in the tandem repeats o We have 280 copies of this o Each transcribed as a single unit o Then processed down to the useful units The 5S tandem repeat is on chromosome 1 and are not transcribed by RNA pol I o 5S is transcribed by RNA pol III o This is the exception to what we need to know about RNA polymerase functions o We need to know this RNA POL III TRANSCRIBES 5S o RNA POL I IS DEDICATED TO THE NUCLEOLUS The NORs in the Micrograph Page 2 of 6 weber uiuc edu 15 October 2007 MCB150 Lecture 22 o Three copies of the 45S gene one after another o It is NOT a 5 8S 18S 28S gene one after another o Rather it is three 45S genes Electron Microscopy Three distinct regions of the nucleolus o Fibrillar regions or centers the regions containing the DNA that is being transcribed o Dense fibrillar regions regions of dense transcription factors and RNA polymerases o Granular zones the bulk of the rest of the nucleolus Final assembly of ribosomal subunits and pre ribosomal particles What is going on in the three Zones Transcription processing assembly Nucleus is separated by a double membrane envelope The dotted line is the boundary of the nucleolus It isn t a separate organelle No membrane envelope It is just a region NORs within the nucleolus o DNA brought together like fingers The fibrillar center o Long strands of rDNA DNA coding for ribosomal material o Basically a region of unwound DNA ready for transcription Dense fibrillar region o DNA is latched on with TFs and RNA polymerases o We are bringing in proteins and enzymes o Transcription moves along o Processing enzymes begin cutting up the rRNA to make subunits o It is dense because there is a lot of stuff happening in the region transcription cutting cleaving protein association etc Granular zone o Assemble ribosomal subunits and pre ribosomal particles o Subunits packaged up Put final touches then it is sent out into the cytoplasm as finished subunits Note the 5S rRNA is not transcribed in the nucleolus o It is most accurately described as being transcribed in wherever chromosome 1 s territory is o All tandem repeats are on chromosome 1 o Wherever chromosome 1 has set up shop is where 5S is transcribed Ribosomal proteins o Have about 80 of ribosomal proteins They were synthesized in the cytoplasm where all proteins are synthesized They came from translation of mRNA The mRNA was exported by the nucleus o Know a ribosome is a complex of protein and rRNA o Know the basic pattern On some other chromosome there was gene X It was transcribed into mRNA exported into the cytoplasm where an Page 3 of 6 weber uiuc edu 15 October 2007 MCB150 Lecture 22 existing ribosome makes the protein that will be hauled into the nucleolar region where it is associated with the next ribosome o Don t worry about the chicken and egg issue here of how a ribosome could be created without having an