CHAP24Biochem - Professor Gracz Chapter 24 notes PDF

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Professor Gracz Chapter 24 notes...

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CHAP 24 – TRANSCRIPTION/ POST – TRANSCRIPTION *** = mentioned in class that we have to know

Transcription = taking genetic information and making it into an expressed product. It is a DNA template-directed biosynthesis of RNA in which one strand of DNA is used to make RNA. 

DNA dependent RNA polymerase makes RNA.

Operon Model = cluster of structural genes in which its transcription is controlled by one operator that either allows or prevents transcription. Bacteria (Prokaryotes) tend to have operon system  

Regulatory gene = can bind to operator and inhibit transcription of the adjacent structural genes. Polycistronic = 3 genes expressed on a single chromosome controlled by a single operator.

Controllers 

Activating genes (Activators) = induces the expression of gene by binding sequence-specifically to a DNA site located near a promoter and making protein–protein interactions with the general transcription machinery (RNA polymerase and general transcription factors), causing the facilitation of the binding of the general transcription machinery to the promoter.

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Factor Proteins = proteins that can bind to nucleotide sequences a) Inducer = binds to repressor and induces RNA polymerase to make mRNA b) Repressor = RNA-binding protein that inhibits the expression of one or more genes by binding to the operator site. Blocks the attachment of RNA polymerase to the promoter, thus preventing transcription

Sucrose-Gradient Technique = technique used for separating RNA. In the tube, the larger the molecule is, the further down the molecule will in the gradient. Found that rRNA has 4S, 16S, and 23S. (S=Svedberg units for sedimentation coefficient). Pulse labeling technique = technique that shows that there are a collection of mRNAs of various sizes by showing how the radioactivity of the labels are distributed heterogeneously. Hershey- Chase Experiment = were a series of experiments conducted in 1952 by Alfred Hershey and Martha Chase that helped to confirm that DNA is genetic material. In their experiments, Hershey and Chase showed that when bacteriophages, which are composed of DNA and protein, infect bacteria, their DNA enters the host bacterial cell, but most of their protein does not Rifampicin ( RNA polymerase inhibitor) = treats bacteria by blocking the synthesis of tRNA, rRNA, and mRNA, indicating that bacteria contain one RNA polymerase (RNAP)

RNA Polymerases = Prokaryotes (bacteria) have just one polymerase. Eukaryotes have 3 RNA polymerases

RNA Polymerase = enzyme that catalyzes transcription. It initiates and extends the polynucleotide chain. It catalyzes the formation of the phosphodiester bond. It brings the complementary nucleoside 5’ triphosphate onto the DNA template. The 3’ OH group then nucleophilic attacks the phosphate group, forming the bond. A pyrophosphate group is released in the process.   

Requires : NTPS, DNA template, Divalent Metal Atom (increases efficiency). DOES NOT NEED A PRIMER TO INITIATE PROCESS Pausing and backtracking control transcription rate and fidelity.

*** Structure of RNA Polymerase = RNA Pol is a holoenzyme with 5 subunits. Weighs 450,000 Da. α subunit = involved in chain initiation and interacts with regulatory proteins and upstream promoter elements. Binds to regulatory sites, such as repressors or enhances. that are far away from promoter sites. UP sites = alpha subunit bound to regulatory sites. β subunit = involved in chain initiation and elongation. Polymerization process is here β’ subunit = involved in DNA Binding. Polymerization process is here. σ subunit = involved in Promoter recognition and promotes transcription ω subunit = involved in the promotion of enzyme assembly. Helps to increase binding of polymerase

σ subunit = determines where the start site of transcription is going to be by recognizing promoter elements (sequence of nucleotides close to the start sit (at +1) ). Recognizes the promoter site at the “Tata” sequence. The Greater the affinity of the σ subunit to the consensus sequence, the greater the efficiency of transcription Promoter sites = Ideally, they are A-T rich, making it easy to open strands. There are two promoter sites. The -35 and -10 promoter sites are consensus sequences, where the σ subunit binds to the gene. Consensus sequences= regions containing similar nucleotides. They indicate the nucleotides that are most of often found at each position. Movement of Polymerase = first, the alpha subunit interacts with promotor elements by binding to upstream elements. This allows the sigma subunit to recognize and bind to the promoter site. Then both Beta subunits encompasses the whole start site. Lastly, DNA opens up causing transcription. DNA Footprinting Analysis = identifies RNA polymerase binding sites on DNA from fragments.

3 Major features of Prokaryotic Transcription : Initiation, Elongation, Termination 1) Initiation = RNA Polymerase (highly processive, doesn’t fall off easily) recognizes the -35 and -10 regions on the DNA strand and binds to it due to sigma subunit. Because the DNA strand is still double stranded, a closed-complex forms. The complex opens at the -10 region where DNA is unwound. A magnesium dependent isomerization further unwinds the DNA from -12 to +2. 2) Elongation = Occurs in the transcription bubble where theres always around 14 unpaired nucleotides. Bubble is caused by opening of the strand. First, chain growth begins by binding incoming template specific NTP, followed by binding of the second. Then nucleophilic attack by 3’OH of the first nucleotide on the a-phosphate of the second nucleotide forms a phosphodiester bond in a manner similar to DNA replication. DNA continuously unwinds in the transcription bubble. Lastly, the sigma factor dissociates during incorporation of the first 10 nucleotides. 3) Termination : Factor Independent / Factor dependent a) Factor Independent = Two symmetrical G-C segments form a stem loop. This formation is caused by transcription base pairing with itself. If RNA isn’t able to base pair with the DNA, transcription won’t happen. So the stem loop weakens the protein DNA-RNA complex. The A-rich segment that follows further weakens these interactions, causing the release of the transcript and termination of transcription. b) Factor Dependent (Rho-factor dependent termination) = Rho protein recognizes a sequence of nucleotides on the DNA-RNA and binds to it. Once its binded it starts moving from the 5’ to 3’ direction, toward the 3’end. It moves faster than the rate in which the polymerase makes the transcript. Once it catches up with the polymerase, the Rho protein disrupts the interactions between the template and the transcript, causing the template and and transcript to dissociate. Rho and polymerase also dissociate.

Eukaryotic Transcription : uses 3 Polymerases that require transcription factors. RNA Pol I = transcribes (rRNA) / uses TF I RNA Pol II = transcribes (mRNA + small rRNA) / uses TF II RNA Pol III = transcribes tRNA / TF III TFIIIA = a zinc finger protein that has alpha helices with histadine and cysteine residues.

Eukaryotes have promoter regulatory elements Tata Box is the eukaryotic version of the -10 region There also may be enhancer or silencer regions upstream of the initiation site.

Eukaryotic Initiation 1) DNA looping brings activator proteins into contact with transcription factors. 2) TBP (Tata Box Binding Protein) binds to DNA, opens it up by bending it 90 ◦ and enhances transcription. 3) TFIIB (has B-linker element), opens up DNA some more Mediator = allows for efficient transcription by dictating communication between enhancer regions and proteins. Bound at the promoter site. High levels of histone acetylation = high transcriptional activity. Acetylation = opens up the DNA due to the weakening of the ionic interactions caused by making the histones less basic. Increases transcription Deactylation = makes histones more basic / decreases transcription

Eukaryotic Termination 1) AAUAAA consensus sequence forms in the primary transcript (pre-mRNA) (the entire gene being transcribed). The primary transcript also has introns and exons. 2) Transcription still continues after the primary transcript is transcribed 3) RNase (Endonuclease) recognizes the AAUAAA sequence on the pre-mRNA and cuts the phosphodiester bond in the DNA – RNA complex that’s 11-30 nucleotides away, and a fragment dissociates. This leaves the transcript with the AAUAAA sequence ending in a 3’OH. 4) Poly Adenylase (type of RNA polymerase) puts on a polymer of adenosines. It does this by recognizing the site and finds the 3’ end, uses ATP, and extends the 3’end with a string of A’s. Forming the Poly A tail (made of 100-300 A residues). 5) We are left with MATURE mRNA at its 3’end always has a string of A’s.

Post Transcriptional events : all are done in the nucleus

1) Capping = all eukaryotic mRNA is capped at the 5’ end by a 7- methylguanosine. i. 1st nucleotide always methylation. 2nd nucleotide may or may not be methylated 2) Polyadenylation = part of how we terminate RNA synthesis. Termination happens when the endonuclease chops of a nucleotide fragment at the 3’end. 3) Splicing = removing any intron sequences that was present in the primary transcript....