Molecuer bio

• Translation basics – process by which mRNA is translated into an amino acid sequence using a ribosome (rRNAs and proteins) and tRNAs.

• Understand the general structure of an amino acid, the peptide bond, and the genetic code (that three nucleotides make a codon and specify an amino acid). How does the tRNA interact with the codon? How does a tRNA become charged with an amino acid? What are the A, P, and E sites in a ribosome, and what happens at each of these three sites?

• Initiating translation in prokaryotes: the small subunit binds the Shine-Dalgarno sequence, the initiator met binds, then the large subunit binds. The first AUG after the Shine Dalgarno sequence is the start codon. What do factors EF-T and EF-G do? Release factors recognize the stop codons, and allow the ribosome to dissociate.

• What is a polysome?

• Why are bacteria able to couple transcription and translation (how can transcription and translation of the same mRNA occur at the same time)? Can this happen in eukaryotes?

• Eukaryotic translation does not use a Shine Dalgarno sequence; instead initiation complex forms at the 5'cap, and the first AUG after the cap becomes the start site. Elongation factors also help bring in the next tRNA and move the tRNAs from the A and P sites to the P and E sites. Does translation end the same way for both prokaryotes and eukaryotes?

• What is a tmRNA and how does it ‘unstick’ stalled ribosomes in prokaryotes?

• How do prokaryotes and eukaryotes slow/stop protein synthesis?

• Post-translational modifications to amino acids increase the type of functions and activities the proteins can have. Identify three types of post-translational modification.

• Chaperonins assist in protein folding. Understand the difference between those that "hold" the protein and those that "fold" the protein.

• Sequences within protein sequence can (1) get the protein into the correct location for its function and (2) tag the protein for degradation. Signal sequences are usually located at the N’-terminus of proteins – what do they cause to happen? Ubiquitin tags have a very different purpose – what will happen to a protein labeled with ubiquitin?

• Understand the differences between primary, secondary, tertiary, and quaternary structure. For example, where are H-bonds (H=hydrogen) relevant? How do secondary and tertiary structure differ? Do all proteins have quaternary structure? For hydrogen bonds, understand how they participate in alpha helices and beta sheets. How do the different R-groups on amino acids contribute to the overall conformation of the protein, and how do they participate in tertiary structure?

• Describe how subunits can assemble (what types of amino acids are you likely to find on the interacting surfaces?) and how interactions in one domain can affect the structure and activity of another domain in the same protein (think about transcription factors).

• Proteins can have many roles; our major focus will be on enzymes. Define "active site", and describe what about the active site determines an enzyme's specificity. Understand what activation energy is and how enzymes can lower the activation energy. What is a substrate analog, does it always resemble the substrate/does it always bind at the active site? Can it be an inhibitor?

• What is an allosteric enzyme? How can it be regulated?

• Now, thinking about the examples of DNA-proteins interactions shown in class, where are the following motifs seen (as in, do they directly participate in protein-protein interactions or do they interact with DNA?): Helix-turn-helix, Leucine zipper (interacting surfaces, see above), and Zn finger.

• Denaturants -- how does SDS cause proteins to unfold? What do urea and guanidinium disrupt?

The proteome is all proteins in a cell encoded by the genome; proteomics is the study of these proteins and their activities.

Before we can study them, we need to get the proteins out of the cell – so, break the cells either manually (blender, sonicator, glass beads), with detergents (these remove cell membranes) or cycles of freeze/thaw. Then, …

Proteins can be separated based size and/or charge (polyacrilimide gels and column chromatography). Note that unlike nucleic acids, amino acids do not have the same charge because the amino acids have the different R groups. Therefore, SDS is used to help to denature proteins and to give them a net negative charge.

How can we pick out a specific protein after separating samples on a gel? Antibodies and western blots -- note that this will give us the protein's location on the blot and if it's in a sample. Understand how an antibody recognizes a specific protein, and how Western blots are the protein equivalent of a Southern blot (DNA) or a Northern blot (RNA).

Mass spectroscopy (MALDI-TOF and electrospray) allows for the precise measurement of the molecular weight of the protein, and from the molecular weight, we can determine the identity of the protein itself. 

Protein tags (MBP, His-tag, FLAG-tag, Strep) can be added to proteins by cloning the coding sequence with the tag sequence. The two get expressed linked together, and now there’s a handy “tag” on your protein of interest that allows you to isolate it easily.

Phage display allows us to screen for particular proteins based on function – does the protein bind another specific protein, does it interact with RNA?. The power of this technique comes from being able to screen for a function, and then have the coding sequence for proteins with that function.

Yeast 2-Hybrid is a large scale screening methods that can identify interactions between proteins. Understand the basic premise of this method -- that if two proteins interact, they form an intact transcription factor and turn on transcription of a reporter gene. The gene coding sequence of expressed proteins are cloned into a vector with the DNA binding domain of a transcription factor; this cloning is repeated for the vector with the transcription factor's activator domain. Yeast strains carrying the two separate vectors are mated (this creates cells with both vectors in them), and if the two proteins interact and form the intact transcription factor, the mated cells will transcribe, and then translate, the reporter gene. How is this reporter gene detected? How can the results of yeast 2-hybrid be confirmed?

Co-Immunoprecipitation (or co-IP). First understand how immunoprecipitation works, in that an antibody is added to a cell lysate (or other sample with proteins in it) and recognizes/binds to its epitope on a protein. This interaction makes for a 'heavy' complex that can be isolated by centrifuging the sample (hence, precipitate. Beads with protein A can also be added to help with the isolation). Anything that interacts with the protein that the antibody binds will get precipitated, too. Understand how we see what is precipitated or pulled-down. For the Co-IP part, this confirms that two proteins are in the same precipitated complex by running immunoprecipitations in parallel, and then checking if the other protein was also in the precipitate.

Cells need to be able to respond to changes in their environment, and they do this by switching genes on and off. How they do this and when they do this depends on what type of response is needed – is the cell going for efficiency (want to save energy/resources), or does it need to react quickly? Understand that by regulating transcription, the cell is being more efficient (making and processing mRNA is expensive!) but takes a longer time to respond.

This chapter focuses on where transcription can be regulated, so with each process, see if it can fit into one of the following categories:

• Access to DNA

• Recognition of the promoter by the polymerase

• Initiation

• Elongation

• Termination

• Understand how the activity of alternative sigma factors can be regulated by their presence (they remain in high temperatures but are degraded at normal temps), by cascades (need multiple factors to activate in a correct series), and binding of anti-sigma and anti-anti-sigma factors.

• Describe the differences between activators (positive regulation) and repressors (negative regulation) in terms of what happens when they bind the signal molecule (do they bind DNA or fall off?), where they bind DNA, and what effect their binding to DNA has on RNA polymerase.

• In prokaryotic genes, it’s common for a cluster of genes with related functions to be transcribed by the same promoter – these are operons. For the lac operon, know what binding sites are in the regulatory region, and how transcription is effected by what is bound to the regulatory region (for the full story, pull out information about glucose and lactose).

• The same DNA binding proteins can act as either activators or repressors, depending on their conformation. For AraC bound to arabinose, it allows RNA polymerase to bind and transcribe. Without arabinose, AraC has another conformation that binds to different sequences on DNA and forms a loop – thus blocking RNA polymerase from binding. Therefore, the shape of DNA binding proteins is critically important in determining where it can bind DNA and what effect it has on transcription.

• Besides signal molecules regulating the activity of repressor, co-repressors (ArgR example) and sequestering proteins (PtsG example) influence the activity of repressors.

• Describe the difference between specific and global control of regulation.

• Eukaryotic transcription has its own special ways of being regulated. With methylation, be sure to know WHAT is being regulated as this can have very different results. On slide 6 we saw that insulator regions could be methylated -- this blocks insulator binding proteins from binding, and lets enhancer activate transcription of genes that are very far away. Then on slide 15, histones can be methylated, too -- and this leads to activation or repression, depending where the histone is methylated. Last, we talked about gene sequences/promoters being methylated -- this silences/turns off genes. So it's important to note what is methylated, and from there you can figure out what the effect of methylation will be. Now for the rest of the learning objectives:

• As we seen with other processes, regulating eukaryotic transcription is more complex than regulating prokaryotic transcription. First, there are more genes. Then the activator sequences can be anywhere (not limited to upstream promoter regions), the genes are contained in the nucleus (transcription factors move between the cytoplasm and the nucleus), and DNA is wrapped around nucleosomes with varying amounts of compaction. Understand that transcription factors recognize and bind to a specific DNA sequence (go over hybrid transcription factor expt on slide 3), and that this binding is required for activating transcription.

• Multiple transcription factors and repressors can bind and effect transcription of a particular gene. What is the role of the mediator complex on eukaryotic transcription?

• Insulators and matrix attachment regions create loops of DNA , which can affect what sequences are near each other physically and can regulate transcription of a gene. How does an insulator restrict the activity/reach of an enhancer? How does methylation of an insulator effect its function?

• Negative regulation in eukaryotes is different from negative regulation in prokaryotes. In eukaryotes, these repressors block the binding of activators to the promoter by physically blocking by binding to the same sequence or dimerizing with a binding protein, but not having a DNA-binding domain itself.

• Understand the physical difference between heterochromatin and euchromatin, and how that affects being able to transcribe from a gene. How does the addition of an acetyl group to histones affect transcription? How does sliding or remodeling of nucleosomes affect transcription?

• What do HATs and HDACs do?

• Methylation of DNA regulates many processes in the cell – does methylation of DNA increase or decrease expression? Understand how methylation of DNA and acetylation of histones work together to turn off transcription. Describe imprinting and how methylation has a role in it. Methylation is also part of X-inactivation.