BMB 462 Lecture 30 Outline of Last Lecture I Comparing transcription and replication II Transcription in bacteria the components involved III The initiation phase IV Sigma factor recognition of promoters V Amino Acid interactions with DNA VI Sigma factor structure mechanism VII DNase I footprinting VIII Initiation and elongation IX Termination Outline of Current Lecture I II III IV V VI dependent termination Differences between eukaryotic and bacterial transcription RNA Polymerase I RNA Polymerase II RNA Polymerase III Transcription inhibitors Current Lecture Concepts to remembers from previous courses lectures There are 2 types of transcription termination in bacteria independent covered last lecture and dependent I dependent termination a dependent transcription termination relies on the protein b It involves pausing of the RNA polymerase near the termination site often in a GC rich sequence which causes the polymerase to slow down because it s harder to melt due to the 3 H bonds c The protein binds to a CA rich sequence in the nascent RNA and then moves in the 5 to 3 direction along the RNA hydrolyzing ATP i The protein has ATPase activity as well as ATP dependent RNA DNA helicase activity that pulls apart the RNA DNA helix formed in the These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute transcription bubble between the newly synthesized RNA and the DNA template strand 1 This pulls the RNA out of the polymerase terminating transcription d Bacterial genes are either going to have a independent terminator that has a special sequence of inverted repeats followed by a run of T s that can easily be recognized in a genome sequence of bacteria just by looking at the sequence e Or they ll have dependent terminators that are harder to recognize f These terminators occur downstream of the gene operon II Differences between eukaryotic and bacterial transcription a Comparing Transcription in Eukaryotes and Prokaryotes i Eukaryotic transcription is separated from translation in space and time transcription and translation are coupled in bacteria RNA polymerase in the transcription bubble is making an RNA As the RNA is being transcribed ribosomes attach to the mRNA and translate a polypeptide ii Coupled transcription and translation is beneficial There are many RNases in cells that degrades naked single stranded RNA covering the RNA with ribosomes protects it to some extent The coupled processes also increases the rate of transcription the ribosomes by translating RNA and bumping up against the RNA polymerase exert a force on the RNA that helps the RNA polymerase go faster b Compartmentalization and increased complexity in eukaryotes results in transcription occurring in the nucleus surrounded by a membrane The mRNA must also be extensively processed to become a mature mRNA this is also done in the nucleus The RNA then needs to be transported out to the cytosol for translation into proteins i Separating transcription and translation by time and space in this manner allows extra opportunities for regulation c A second difference between eukaryotic and bacterial transcription is there is more inhibition by packaging in eukaryotes i DNA is highly compacted in nucleosomes fibers and other higher order structures The extent to which these formations need to be unpacked to allow transcription is unknown but it is clear it offers regulation 1 The nucleosomes hold DNA in such a way that represses gene expression keeping the genes inactive in the chromatin a To overcome this activation of transcription in eukaryotes is very common In particular the promoter region in active genes is not wrapped in nucleosomes if they aren t nucleosome free the cell needs to bring in a transcriptional activator and a nucleosome remodeling factor to remove the nucleosome covering the promoter b This allows access for other general transcription factors that recruit the RNA polymerase to the promoter 2 This is regulated by chromatin modification and remodeling factors such as histones loosen the grip of nucleosomes on the DNA and allow other factors to bind 3 Nucleosomes don t have to be completely removed RNA polymerase is capable of elongating through nucleosomes without completely dissociating them d The 3rd difference i There are multiple distinct RNA polymerases in eukaryotes polymerases I II and III 1 All 3 are composed of around 13 subunits and all can be found in the nucleus Polymerase I though is found in a separate compartment of the nucleus called the nucleolus where most of the rRNA is made ii Polymerase I makes 3 of the 4 rRNAs in eukaryotes polymerase III makes the fourth iii Polymerase II makes the pre mRNAs it transcribes the protein encoding genes 1 Polymerase II makes up 20 40 of the relative polymerase activity in the cell even though the mRNA it transcribes only amounts to 5 of the total RNA This is because the pre mRNAs are very large due to the introns they contain These introns are spliced out as the mRNA is transcribed a Thus a lot of energy is wasted in processing these premRNA aka heterogeneous RNA or hnRNA due to the fact they are heterogeneous in size they come in all different sizes and the size changes very rapidly as introns are spliced out iv Polymerase III accounts for the smallest portion of relative activity as it makes the small tRNA and the smallest rRNA v amanitin is an inhibitor of polymerase activity but inhibits differentially between polymerases I II and III Inhibition of the various polymerases allowed scientists to study the relative activity of each one 1 amanitin innhibits polymerase II very strongly By adding excess amanitin it is eventually able to inhibit polymerase III activity Because polymerase I is not influenced by amanitin addition of the inhibitor leaves only polymerase I activity e The fourth difference i Eukaryotic RNA polymerases only bind to promoters after general transcription factors and sometimes specific transcription factors have bound 1 Unlike the bacterial polymerase holoenzyme which recognizes the promoter via the sigma subunit eukaryotic general transcription factors recognize and bind to the promoter and then the polymerase recognizes the proteins III RNA Polymerase I a Polymerase I function is the simplest of the 3 b There is only one polymerase I promoter which is repeated many times In eukaryotic genomes the 3 genes that are going to be
View Full Document
Unlocking...