* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download doc NTC Mar 31
Community fingerprinting wikipedia , lookup
Protein moonlighting wikipedia , lookup
Molecular cloning wikipedia , lookup
RNA silencing wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Messenger RNA wikipedia , lookup
List of types of proteins wikipedia , lookup
Polyadenylation wikipedia , lookup
Biochemistry wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular evolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Gene regulatory network wikipedia , lookup
Point mutation wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Epitranscriptome wikipedia , lookup
Non-coding RNA wikipedia , lookup
RNA polymerase II holoenzyme wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Gene expression wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Lecture 34: March 31st Gene Regulation I- Prokaryotic gene regulation II Summary of where we ended last time: Went through experimental approach to the negative regulation of the lac operon We know that this inducible and that it is negative control So if lactose is not present in the culture medium then the Laci gene is transcribed and is able to bind into the laci repressor protein, which is able to bind to the operator which is a DNA sequence upstream of the promoter of the Lac Operon. Therefore the RNA polymerase has a physical block, so no proteins are produced there When lactose is present the Laci protein can interact with lactose, it undergoes an allosteric change, which makes it no longer able to bind to DNA so the operator sequence is now free, and can be transcribed by the RNA polymerase, to be translated into proteins utilized for the breakdown of lactose The function of Beta Galactosidase and Permase is known, and the function of transacetylase is still unknown If the permase is needed to uptake lactose, how come the organism can detect lactose? Because there is a low level of transcriptional activity, allowing it to sense lactose. Bacterial growth responds to the presence of difference sugars: A lot of primary work on understanding the lac operon was done using growth curves You look at how many bacteria are present in the growth medium over time, and if the bacteria are able to grow and divide, the number will increase over time If you grow bacteria in a rich medium, containing glucose, you will see, no matter what bacteria you use, you will see different phases Lag phase: initial slow phase, the bacteria are getting used to the medium Log phase: Dividing regularly at intervals of about twenty minutes, and over time of population doubles Stationary Phase: At some point the medium is not as rich as it was initially because the bacteria have been growing and producing catabolites, so growth begins to slow, there is no more division and the cells are alive Death Phase: all of the nutrients have been consumed, and the number of cells declines They were interested in different enzyme activations, so they started to mix various 1 Lecture 34: March 31st sugars, similar curves could also be obtained with different sugars, as long as there is only one kind in the medium So he tried mixing various sugars If you combine glucose and lactose Growth curve becomes bi-phasic In phase I cells use glucose In phase II, glucose is exhausted, and they change to using lactose, creating a second logarithmic phase So this means that bacteria can sense and choose between different sugars He started to study the physiology behind this situation Showed him - Positive control of the lac operon Glucose is the preferred energy source, so when both glucose and lactose are available the operons for sugars other than glucose are off There is always a very low transcription in the cells There is balance in the cells not to transcribe and translate proteins that are not needed, but have a little bit to allow for adaptation The approach was done through the study of particular mutants Cya and Crp mutants Decreased levels of lacZ and lacY upon induction In lacI-- cya and crp mutants exhibit lower levels of these enzymes Suggests that these two genes are also involved in the regulation of the lac operon However, they map outside the lac operon The cya gene encodes for adenylate cyclase which produces cyclic cAMP, starting from glucoses Crp (cAMP, receptor protein) gene encodes for the catabolic activator protein (CAP) that binds cAMP These two genes interact with each other Later on, cyclic AMP and its function were studied elsewhere and it was found that cAMP levels are regulated by the amount of glucose in the cell They are inversely regulated (low glucose = high cAMP) When there is high cAMP, CAP protein can bind with it, there is an induction of an allosteric change and this protein can bind to the lac promoter, positively activating it What happens on the molecular level: When CAP binds to DNA Structural studies have shown that when CAP binds to DNA it wraps the DNA 2 Lecture 34: March 31st around itself The DNA is stretched and bent, exposing the nucleotide sequence much better, so the RNA polymerase can recognize it, and bind it more efficiently In addition to this, it has been shown that RNA polymerase and the CAP protein can interact with each other, so RNA is also stabilized in the proximity of the promoter The Lac repressor and CAP proteins bind to DNA sequences with two-fold rotational symmetry Certain nucleotide positions are more important than others (these are highlighted in the figure) If you look at the polarities, the sequences are sort of palindromes, and in the center of the symmetries is the dot (highlighted in red) They are multimers- you do not have one molecule that binds to the DNA, but they interact with each other, they are either dimers or tertamers There is an advantage in this type of organization because it docks two molecules and is stronger binding than just the single protein The other observation is that the AT pair is thermodynamically weaker than GC (AT only has two hydrogen bonds so takes less energy to open up the helix) When we look at these two sequences that there is more AT pairs than GC pairs, in the lac operator so it is thermodynamically easier for RNA polymerase to open this region One possibility is that the bending facilitates the opening of the two DNA strands The Lac Operon at the molecular level: 3 Lecture 34: March 31st Shows all the different binding sites upstream of the lac operon The lacI gene: synthesizes the repressor all the time Then you have a series of binding sites that are partially overlapping The site where CAP binds once it complexed with cyclic AMP Site where that RNA polymerases recognizes Then there is the operator, which binds the repressor The transcription start is overlapping this region Then there is the Shine-Dalgarno sequence which promotes mRNA translation There are a series of regulatory requirements that are next to each. The proximity makes them work together They can work together- eg. Cap protein facilitates RNA binding It can be exclusive, eg. The repressor binding site stops all transcription, blocking the RNA start site What does this mean in terms of bacterial physiology Glucose present: cAMP is low, no cap protein complexed , no lactose is metabolized, no lac mRNA is synthesized Leaky expression Glucose and lactose present: They system is balancing, there is not a lot of cAMP, but there is a bit of lactose metabolized Depends on the relative concentration of glucose, how much cAMP is there Occasional mRNA No glucose, and lactose is present CAMP high, all of the enzymes to metabolize lactose In bi-phasic growth, the first plateau is when the bacteria is synthesizing all the new enzymes This examples shouws that positive and negative regulation can work in the same time Why use both Is would be simpler, but it would not be as adaptable More advantageous for evolution The Ara operon: A lot of similar features to the lac operon 4 Lecture 34: March 31st Similarities with regulation of lac-operon Presence of cis regulatory sequences, Genes are arranged together in operating units Depends on regulatory genes and proteins to be expressed, these regulatory genes and proteins undergo allosteric changes Also uses cAMP-CAP complex Positive control of Ara The initiator of the process is AraC, situated early in the operon When there is arabinose present, AraC undergoes an allosteric change and binds the DNA, activating the operon The cAMP-CAP complex also stimulates transcription Negative control of Ara ( in the absence of arabinose) AraC has a different conformation, changes its properties and makes it behave as a repressor AraC can still bind to the AraI, but it can also bind to AraO The result of this double interaction with the DNA is that the DNA is bent Steric impediment for mRNA transcription to begin Summary: Activating and repressing mechanisms coexist and the can cooperate Regulatory proteins undergo allosteric changes upon binding affector molecules and result in different protein properties Activating effector proteins bind to DNA and may change DNA conformation to help recruit the RNA polymerase complex Activators may directly interact with RNA polymerase and further stabilize the transcription initiation complex Repressors hinder the recruitment of RNA polymerase complex to the transcription site by various mechanisms (steric hindrance, conformational change of DNA etc.) 5