Rationalize the difference in boiling points for each of the following pairs of substances: a. n-pentane CH3CH2CH2CH2CH3 36.2C b. HF 20C HCl 85C c. HCl 85C LiCl 1360C d. n-pentane CH3CH2CH2CH2CH3 36.2C n-hexane CH3CH2CH2CH2CH2CH3 69C 38. C
BCHM EXAM 3 WEEK 2 NOTES 3/2/163/11/16 3/2/16 Repression and Depression Negative Control Genes are ALWAYS transcribed unless a repressor is present. + Lactose Lactose Transcription No transcription Derepresesed Repressed Repressor is not bound Repressor is bound to the operator Repressor is bound to the operator Activation v DeactivationPositive control genes are not transcribed until an activator is present + Glucose Glucose No cAMP= no cap Increase in adenylation No activation Increases cAMP= Increase in CAP you cannot have CAP without cAMP. These molecules bind to the CAP protein. REPRESION AND DEREPRESSION RELYS ON LACTOSE ACTIVAITON AND DEACTIVATION RELYS ON GLUCOSE +Glu +Glu Glu Glu +Lac Lac +Lac Lac Production + +++ (50 times /+ as much) Repressor Bound to Bound to Bound to Bound to allolactose operator lactose operator cAMP and None None Increase cAMP Increase cAMP CAP and CAP and CAP o The big picture of all this mess is that the bacteria wants glucose. Wants it bad. So the bacteria knows that breaking down lactose will give you glucose. Therefore, when glucose levels are low and lactose is high, the lac operon is like LETS GO! This concept explains all of the other scenarios in the table above. o This also explains why it is an inducible operon! TRP operon o Synthesize trp (OPPOSITE OF LAC OPERON) (ONLY HAVE TO KNOW THIS) o Repressible Transcriptional Regulation Epigenetic Regulation (DNA modification) o Addition of methyl groups to cytosine (methylated) Happens in CpG islands It is associated with reduced transcription Methylated DNA can change the way the DNA is read Can be due to diet and stress o Histone modification add acetyl to lysine or arginine loosens DNA and enhances DNA 3/516 Enhancers activators and Mediators o Activator binds to enhancer and stimulates transcription o Mediator proteins mediate interaction between enhancers and DNA o Transcription initiation complex: TF, Activators, RNA poly II and mediator o LCR Locus Control Region Example of enhancer region in DNA Depressors and Silencers o Repressors bind to silencers o Inhibits DNA o You need to know this picture o Transcription Posttranscription (Eu)(Hat) Acetylation of histones Hetero Deacylation of histones Alternate splicing HDAC Methylation(hetero) with miRNA and siRNA dimethyl (eu) Methylation of cytosine (CPG) Activators, Silencers, mediators Post transcriptional o Removal of introns o Splicesome Enzyme complex snRNP and snRNA’s (RNA interference) can cut it in different ways MicroRNA’s and siRNA’s small repeats that bind to dicer by forming a hairpin and then degraded or block translation Steps of RNA interference The miRNA folds on itself Dicer comes and makes it into smaller fragments miRNA fragments come and bind to the protein complex the protein complex and miRNA binds to the RNA Then it is either degraded (miRNA) or blocks transcription (siRNA) 3/9/16 Details of protein synthesis (translation) requirements for translation o mRNA o Large (50s) and small (30s ) subunits o Initiation and elongation factors o Amino acyl tRNA synthases (only ones that use ATP in this process) o GTP energy for join subunits deposit Amino Acids in A site repositioning Amino Acids (peptide chain to the next tRNA in the A site o Ribosome 3 sites A site P site E site (APE like the animal) Ribosome moves!!! not the mRNA peptidyl transferase (apart of the large ribosomal subunit) o shine dalgarno sequence purine rich mRNA sequence just upstream of AUG start codon Recognition is facilitated by the base paring with the 16s rRNA consensus sequence: AGGAGG don’t have to memorize the sequence Prokaryotic translation: o Initiation: This is the first step when the mRNA strand arrives at the ribosome Formation of the initiation complex The shinedalgarno sequence: this is a purine rich sequence upstream from the AUG codon. Recognition of this sequence is crucial and is recognized by the 16s rRNA strand. o Consensus sequence: AGGAGG Sequence of events: o IF1 and IF 3 bind to the 30 s subunit IF1 blocks the A site until the first tRNA is in place IF3 blocks the 50s subunit from binding the 30s subunit. o 30s subunit binds to the shine dalgarno sequence o 30s subunit slides in place over the AUG start codon (5’3’) o Insure that the first AUG start codon is located o IF2 with GTP binds to the 30s Asite o IF1 is then displaced and the fMettRNA (ONLY PROK) o GTP is hydrolyzed which makes IF2 and 3 fall off o The 50s subunit then comes and binds the complex thus creating the characteristic ribosome o Elongation: The EfTu protein carries the charged tRNAs and then hyrolzes GTP to attach it to the codon in the A site. GTP is then rehposphrylated and the EFtu is then reattached with a charged tRNA. peptidyl transferase (in the large ribosomal subunit)then attahces the growing polypeptide chain to the charged tRNA in the A site. EFGGTP moves the ribosome in the 3’ direction which shifts the tRNA’s over a site. The energy released from the GTP causes the ribosome to change conformation. o Termination: The stop codon calls for the initiation factor which comes and bids to the A site This converts peptidyl transferase into hydrolase which cuts the polypeptide away from the tRNA and Ribosome falls away o Multiple ribosomes bind to the same sequence of mRNA and forming polysomes which amplify the protein. Prokaryotes Eukaryotes Ribosome Subunits 30s+50s=70s 40s+60s=80s RNA processing rRNA’s, tRNA’s rRNA’s,tRNA’s mRNA’s Transcript Recognition Polyg, shine delgarno 5 cap and poly a tail sequence Rate 20aa/sec coupled 1aa/sec not coupled IF 3IF’s 9IF’s Initiator AAtRNA Nformyl methionine Methionine Eukaryotes also have Greater diversity of post More complexity of translational targeting proteins modifications 3/11/16 Antibiotics that block transcription or translation o Prokaryotes only: Tetracycline: blocks the aminoacyl tRNA from binding to the a site Chloamphenicol: blocks the peptidyl transferase on the ribosomes Erythromycin: blocks the translocation of the ribosomes Cordycepin o Eukaryotes only α amanitin blocks transcription of mRNA by binding to RNA Poly II o ON HER POWERPOINT (LECTURE 13) WE ONLY HAVE TO KNOW THE ONES THAT SHE BOXED! Cracking the genetic code Synthetic mRNA Polypeptide UUUUUUU PhePhePhe AAAAAAA LysLysLys CCCCCCCC ProProPro UCUUCUUCU SerLeuSer and LeuSerLeu o So…. the experiment was done by addition a bacterial soup with synthetic mRNA strands. The bacteria soup consisted of everything required for protein synthesis except real life mRNA strands. o By doing a table like this, they worked liked dogs and figured out the amino acid code by running a crap ton of trials! o o Things you should look out for with this table they are all in the 5’ to 3’ direction THESE CODONS ARE ON THE mRNA STRAND AA are encoded by more than one codon AUG= start=met UAA,UAG, UGA= stop Mutations RNA polymerase can fix errors But it is not as detrimental as an error in DNA replication because in DNA replication, if you make a mistake, then all of the proteins that come from that DNA I’ll be wrong. Types of mutations Translation mutations: o Missense: This is where an incorrect Amino acid is inserted into the protein sequence. This can be detrimental as it can influence misfolding of the protein and thus altering its function o Nonsense: This is a mutation where a stop codon is sequenced and causes early termination of translation. Usually results in dysfunctional protein that is useless o Silent: This typically occurs during the wobble hypothesis where the third nucleotide of the codon is switched for another nucleotide. However, to our luck, they code for the same protein and thus the protein goes on about its life. You would not know these occur unless you looked at the genetic code. Transcription mutations: o Frame shift mutations: can be an insertion or a deletion that cause the frame of reading (codon) to be shifted over or back one Insertion: This is when a n extra nucleotide is inserted into the mRNa strand that cause the frame to be moved over in the 3’ direction. Deletion: This is when a nucleotide is deleted and the frame is shifted over to the 5’ direction BE ABLE TO LOOK A STRAND OF mRNA, DNA, OR A POLYPEPTIDE AND KNOW WHAT TYPE OF MUTAITON OCCURRED!!!!!