BMB 462 Lecture 30 Outline of Last Lecture I. Comparing transcription and replicationII. Transcription in bacteria: the components involvedIII. The initiation phaseIV. Sigma factor recognition of promotersV. Amino Acid interactions with DNAVI. Sigma factor structure/mechanismVII. DNase I footprintingVIII. Initiation and elongationIX. TerminationOutline of Current Lecture I. ρ-dependent terminationII. Differences between eukaryotic and bacterial transcriptionIII. RNA Polymerase IIV. RNA Polymerase IIV. RNA Polymerase IIIVI. Transcription inhibitorsCurrent LectureConcepts to remembers from previous courses/lectures:- There are 2 types of transcription termination in bacteria; ρ-independent (covered last lecture) and ρ-dependentI. ρ-dependent terminationa. ρ-dependent transcription termination relies on the protein ρ.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-bondsc. The ρ protein binds to a CA-rich sequence in the nascent RNA and then moves in the 5' to 3' direction along the RNA, hydrolyzing ATPi. The ρ 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 ρ-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 ρ-dependent terminators that are harder to recognize.f. These terminators occur downstream of the gene/operon.II. Differences between eukaryotic and bacterial transcriptiona. 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 inthe 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 fasterb. 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 proteinsi. Separating transcription and translation by time and space in this manner allows extra opportunities for regulationc. 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 atranscriptional activator and a nucleosome remodeling factor to remove the nucleosome covering the promoterb. This allows access for other, general transcription factors that recruit the RNA polymerase to the promoter2. 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 withoutcompletely 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 activityin 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 pre-mRNA (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. α-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. α-amanitin innhibits polymerase II very strongly. By adding excess α-amanitin, it is eventually able to inhibit polymerase III activity. Because polymerase I is not influenced by α-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 thepromoter via the sigma subunit, eukaryotic general transcription factors recognize and bind to the promoter and then the polymerase recognizes the proteins.III. RNA Polymerase Ia. Polymerase I function is the
View Full Document
Unlocking...