Lecture 11: Translation Regulation

Regulation of Transcription Side Notes: Genes can be controlled at different levels The RNA polymerase holoenzyme can transcribe any gene that has a functional promoter One level of regulation is the promoter strength, but most regulation comes from targeted gene regulation: Controlling transcription initiation (most prevalent) Controlling elongation or termination Regulation from the transcribed RHA itself Regulatory proteins that decrease the level of transcripts for certain genes are called depressors Proteins that increase transcription are activators Principles of transcriptional regulation Many genes are regulated by proteins that bind near the gene, often upstream of the promoter Regulatory sequences are specific DNA regions to which regulatory proteins bind Regulatory sequences can be directly upstream of the promoter or can be many kbp away In bacteria, sequences recognized by regulators are called operator sites, and are typically close to the promoter or overlapping it If operators are distal to the gene, the DNA must loop around for the regulatory protein to interact with the polymerase, sometimes aided by architectural DNA binding proteins Eukaryotic genes are more often controlled by distal regulatory sequences than bacterial genes - these can be several thousand base pairs away and are called enhancers Regulatory sequences frequently bind several regulatory proteins - this increases the sophistication and subtlety of the regulation Enhancers can be upstream or downstream of the gene As in bacteria, distal sequences regulate gene expression by looping Eukaryotic DNA binding proteins recruit regulatory complexes Transcription factors (TFs) must be able to specifically recognize the correct regulatory sequence Each TF has a DNA-binding domain that recognizes a specific sequence TFs are often modular, and can have addition domains that: Aid oligomerization Activate or repress transcription Interact with other regulators Eukaryotic TFs often control transcription by recruitment of co-activators or co-repressors Co-activators and co-repressors cannot bind DNA alone, but are recruited by specific TFs The helix-turn-helix is a common DNA-binding domain in TFs DNA binding motifs are tailored to fit DNA. Some are found throughout life, whereas others are found just in eukaryotes or metazoans The helix-turn-helix is common and found in many protein folds. It has the first helix and the recognition helix Many are dimers with the recognition helices spaced 3.4 nm apart to fit in neighboring grooves Recognition helices in helix-turn-helix proteins "read" the DNA sequence Regulation of Transcription Side Notes: through interactions with the base pairs One common eukaryotic DNA binding domain is the homeodomain Helix 3 fits in the major groove. Side chains interact with DNA base-pairs The N-terminal arm makes contacts in the minor groove Zinc fingers are the most common human DNA binding domains A zinc finger is a prominent part of many DNA binding folds, and has a domain of ~30 amino acids with an alpha helix and two beta strands around a central zinc ion The zinc interacts with two cysteines and two histidines - these are always conserved in these proteins Proteins often have several zinc fingers, each of which inserts its alpha helix into the major groove Zinc fingers have a flexible DNA-recognition code Zinc finger domains recognize different sequence combinations, depending on their amino acid sequences in the recognition helix Zinc finger TFs contain several domains, each of which will contribute to a longer DNA binding motif DNA binding triggers folding of bZIP TFs Coiled-coils are alpha-helices that wind round each other Coiled-coils form part of some DNA binding domains Basic region-leucine zipper (bZIP) proteins have long alpha helices of about 60 amino acids The two helices have hydrophobic leucine residues on the surfaces that "zip" together. At the N-terminal end, the helices splays and sit in the DNA major groove When not bound to DNA, the basic region is disordered Related proteins like basic region-helix-loop-helix (bHLH) proteins also become structured upon binding to DNA Beta sheets and 100ps can also mediate DNA recognition The MetJ depressor binds DNA with two beta strands p50 and p65, which are part of the mammalian transcriptional regulator nuclear factor (NF)-kB, are largely beta sheet Immunoglobulins are entirely made of beta sheets and connecting loops Trp repressor blocks RNA polymerase binding A simple way to regulate transcription is to prevent RNA polymerase accessing the promoter, such as in the E. coli Trp repressor The Trp repressor is a helix-turn-helix protein. Repressor binding to DNA depends on the level of tryptophan in the cell The several genes needed to synthesize tryptophan are located together, regulated together and transcribed together - an operon When tryptophan levels are low, the genes transcribed, to make tryptophan When tryptophan levels are high, the tryptophan repressor binds to the Trp operator and blocks RNA polymerase from binding The Trp repressor can only bind to DNA when it is itself bound by tryptophan, which only happens when tryptophan levels are high CAP recruits RNA polymerase to promoters Enhancing the binding of RNA polymerase to a promoter can activate Regulation of Transcription Side Notes: transcription Catabolite activator protein (CAP; or cAMP receptor protein - CRP) activated more than 100 E. coli promoters when carbon sources are low Glucose depletion leads to increase in cAMP levels - this binds to CAP and increases its binding to DNA CAP enhances RNA polymerase by binding to the CTD In class I, CAP binds upstream of the promoter In class II, the CAP binding site overlaps the RNA polymerase binding site CAP and the Lacl repressor regulate the lac operon The lac operon responds to low glucose levels in the cell by allowing it to metabolize lactose instead, if lactose is present Lactose binds to the lactose repressor, preventing it from binding When lactose is absent, the repressor binds to the lac operator and transcription is off In low glucose, available lactose conditions, CAP binds to the CAP operator, enhancing transcription, which is strongly on In high glucose, available lactose conditions, CAP does not bind the CAP operator. However, the lac repressor is not bound, so there is weak transcription Activation by NrtC involves DHA looping The RNA polymerase holoenzyme that contains sigma-54 needs to be activated by ATP hydrolysis to drive promoter opening NrtC is an example of one of these activators - it activates genes that are important for nitrogen metabolism NrtC binds at enhancer elements and activates transcription when phosphorylated Phosphorylation triggers oligomerization of NrtC, and promotes interaction with the polymerase at the promoter Oligomerization also stimulates its rate of ATP hydrolysis, which helps promote formation of the open complex Two-component signal transduction pathways Response to external signals often involve phosphorylation of a receptor protein that leads to a signaling cascade One example is a two-component signal transduction pathway Two-component systems have a sensor kinase and a response regulator A histidine in the sensor becomes autophosphorylated on receipt of the signal The phosphoryl group is then passed to an aspartic acid residue in a response regulator protein Lambda phage control of lysis vs. lysogeny Complex and differing signals regulate transcription. The outcome of the combination of signals, which can be antagonistic, depends on the strength of the opposing regulators One example is bacteriophage lambda gene regulation Bacteriophage lambda infects E. coli by injecting its chromosome into the bacterium The phage then enters lytic growth or undergoes lysogeny - the choice depends on host conditions In lysogeny, the virus integrates into the host genome and becomes dormant (the integrated virus is a prophage) - this lets the phage multiply in conditions Regulation of Transcription Side Notes: where there might not be enough nearby cells to infect Lytic growth produces lots of copies of the virus in the bacterial cell and eventually leads to cell lysis - this allows the phage to 'escape' a cell that may not survive Lambda phage control of lysis vs lysogeny With E. coli-containing prophage endures difficult conditions (such as DNA damage), the phage can be triggered to excise itself and undergo lytic growth Lysis and lysogeny are regulated by levels of the DNA binding proteins cl, cll and Cro These bind to sites in the phage chromosome and control transcription at the four promoters P(R), P(L), P(RE), ANF P(RM) The P(R) promoter first produces Cro and cll If there is enough cll, it stimulates transcription at P(RE), which produces cl cl stimulates P(RM), which makes more cl. High cl levels bind to P(R) and P(L) and repress lytic genes, and also activates transcription of integrase Therefore, high cl levels promote and maintain lysogeny If not enough cll is produced, Cro binds to the P(RM) promoter and represses cl production, ensuring expression of the lytic genes Levels of cll are thus crucial to whether the cell enters lysogeny or enters a lytic phase Environmental factors regulate the levels of cll - cll is sensitive to host cell proteases If proteases are highly active, cll is degraded, cl cannot accumulate and the virus enters lytic growth If cll is maintained at high levels, cl accumulates and lysogeny is maintained DNA - binding activities of Cro & cl determine lysis vs lysogeny The binding affinities of cl and Cro at six DNA binding sites determines the choice between lysogeny and lysis cl has two domains that can form tetramers and oligomers. Cro has a single small domain that binds DNA as a dimer The operators (OL and OR) at the PL and PR promoters have three sites that cl and Cro can bind cl binds first to OL1 and OR1 where it has the strongest affinity - this recruits another cl to the weaker sites OL2 and OR2 The cl units oligomerize to form a looped out structure. This stops RNA polymerase binding to PL and PR, and promotes transcription from PRM (leading to increased cl levels) With the increasing levels of cl, it binds to OL3 and OR3, stopping activation of PRM suppressing cl synthesis - cl therefore regulates its own synthesis (autoregulation) If there is insufficient cl, Cro accumulates and binds with greatest affinity to OR3, blocking PRM and suppressing cl synthesis. More cro synthesis leads to binding at the other OL and OR sites, autoregulating Cro Prophage is induced by cl cleavage The prophage must be able to excise itself and switch from lysogeny to lytic growth if the host cell is threatened This is brought about by a response to DNA damage that disables cl DNA damage prompts the host SOS response, activating RecA. RecA cleaves cl so it cannot dimerize or cooperate with other cl units cl then dissociates from operator sites, allowing transcription from PL and PR, Side Notes: allowing lytic growth Lambda N protein prevents transcriptional termination Regulation can also occur at the elongation and termination steps of transcript synthesis Some phage lambda genes are transcribed only when transcription termination is actively prevented - this is called anti-termination Initially, transcription from the PL and PR promoters successfully makes Cro and N, but encounters termination sites tL and tR, terminating the transcripts When N protein builds up, it stops termination N binds to nut sites in the transcribed RNA in a step-loop form. Several proteins are recruited and interact with RNA polymerase, allowing it to transcribe through tL and tR Attenuation controls the effects of terminator sequences Attenuation controls bacterial genes needed for amino acid biosynthesis This exploits folding of the RNA into alternative secondary structures The trp operon is controlled by levels of tryptophan in the cell - different levels cause the transcript to have different secondary structures The genes for tryptophan synthesis are transcribed together (a polycistronic message) The trp mRNA has a leader sequence near the start that has an intrinsic terminator (an attenuator), and two sequences that can form stem-loops This leader sequence has a small open reading frame with lots of codons for tryptophan The leader sequence has 4 blocks (1, 2, 3 and 4) that can form alternative pairing arrangements. 1 and 2 can form a stem-loop, 3 and 4 can form a Rho-independent terminator. 2 and 3 can also form a stem-loop (leaving 1 and 4 unpaired) In low tryptophan, the ribosome stalls when translating the leader peptide region 1 is blocked, so 2 and 3 form a terminator, so transcription continues In high tryptophan, the ribosome proceeds, allowing the terminator to form Riboswitches can control transcription termination Riboswitches are portions of a transcript that can directly bind a small molecule that controls the RNA secondary structure, regulating transcription or translation Riboswitches have two regions - the aptamer that binds to the metabolite, and an expression platform which controls transcription or translation The B. subtilis adenine riboswitch regulates adenine synthesis and transport. Gene expression depends on whether a terminator or ant-terminator forms In low adenine, the RNA structure is as in regions 2 and 3 form an antiterminator and transcription proceeds High adenine allows to form - regions 3 and 4 form a terminator