Transcriptional and Posttranscriptional Regulation Flashcards
Differential gene expression
Although all cells in an organism have the same genes, not all of the genes are expressed in every cell. There is differential expression across cells, with cells synthesizing and accumulating different sets of mRNA and protein molecules
A cell can control the proteins it makes by (6)
- Transcriptional control
- RNA processing control
- RNA transport and localization control
- Translational control
- mRNA degradation control
- Protein activity control
Transcriptional control
When a cell controls when and how often a given gene is transcribed. Ensures that the cell will not synthesize superfluous intermediates
Transcriptional regulators
A group of proteins that determines which genes are transcribed. They recognize cis-regulatory sequences (genetic switches)
Cis-regulatory sequences (genetic switches)
Sequences of less than 20 base pairs that are on the same chromosome (cis) to the genes that they control. Transcriptional regulators undergo complementary binding to these sites. Genetic switches are disperses throughout the genome and can be read without breaking the double helix
Binding between transcriptional regulators and gene switches
Binding is complementary. Consists of many weak, noncovalent contacts that create a strong interaction.
Major groove
A groove in DNA that is wider, where the backbones are farther apart. Since the groove is wider, it displays more molecular features than the minor groove. Nearly all transcription regulators make the majority of their contacts with the major groove
Minor groove
A groove in DNA that is narrower, where the backbones are closer together
Which features of DNA are found in both major and minor grooves?
The outside of the helix is studded with DNA sequence information that transcription factors recognize- the edge of each base pair presents a distinctive pattern of hydrogen bond donors/acceptors and hydrophobic patches
How do transcription regulators recognize genetic switches?
Complementary base pairing
Why must other factors increase the affinity of transcription regulators for DNA?
Sequence-specific DNA binding proteins recognize a range of closely related sequences rather than one specific one. The affinity of the protein for DNA depends on how closely the DNA matches the optimal sequence. However, exact nucleotide sequences may appear randomly throughout the genome. Therefore, there must be more mechanisms to control transcription
Dimerization of transcription regulators
Many transcription regulators form dimers. Both monomers make nearly identical contacts with DNA. Dimerization doubles the length of the genetic switch that is recognized and greatly increases the affinity and the specificity of transcription regulator binding. As a result of dimerization, the DNA sequence recognized by the protein has gone from 6 to 12 base pairs, so there are fewer matching sequences
Zinc finger proteins
These motifs include one or more zinc atoms as structural components. The zinc atom holds an alpha helix and beta sheet found together. These zinc fingers are often found as clusters. The alpha helix contacts the major groove of the DNA and forms a nearly continuous stretch of alpha helices along the groove
Beta-sheet DNA recognition proteins
Structure is a two-stranded beta sheet, so alpha helices are not involved in recognizing DNA. The beta sheet can recognize many different DNA sequences. The bacterial Met repressor is one example of this structure
Leucine zipper proteins
Two alpha helices are joined together to form a short coiled-coil. The alpha helices are held together by interactions between hydrophobic amino acid side chains (usually leucine). Just beyond the dimer interaction, the motif forms a Y-shaped structure, allowing their side chains to contact the major groove of DNA. Results in clothespin-like binding to the major groove
Helix-loop-helix proteins
Consists of a short alpha helix that is connected by a loop to a second, longer alpha helix. Its C terminus recognizes the DNA, while the N terminus helps to position the protein. The loop is flexible, allowing one helix to be against the other and form the dimerization sequence. May be a homodimer or heterodimer. The structure resembles a leucine zipper.
Types of DNA-binding motifs (5)
- Zinc fingers
- Beta-sheet DNA recognition proteins
- Leucine zipper
- Helix-loop-helix
- Peptide-loop motifs
Zinc finger structure
There are several types, but the original motif has a finger-like appearance; its shape depends on zinc binding. Generally defined as motif where α helix binds DNA and zinc serves as structural element
Types of leucine zipper motifs
These motifs can be homodimers (bind to symmetric DNA sequences) or heterodimers (bind different DNA sequences). This greatly expands the repertoire of the DNA switches that can be recognized
Peptide loop motifs
Protruding peptide loops bind DNA instead of α helices and β sheets. p53 functions this way. It recognizes DNA in major and minor grooves to regulate cell growth and proliferation.
Types of gene regulation (2)
- Negative regulation
- Positive regulation
Negative gene regulation
When a bound repressor protein prevents transcription. A ligand binds to remove a regulatory protein from the DNA
Positive gene regulation
When a bound activator protein promotes transcription. The ligand binds to allow the regulatory protein to bind to DNA
Tryptophan repressor
Found in E. coli, switches off genes. In this bacteria, 5 genes code for enzymes that manufacture tryptophan. When tryptophan concentrations are low, the operon is transcribed, and mRNA is translated to produce the enzymes that make tryptophan. When tryptophan is abundant, the amino acid is transported into the cell and shuts down the production of enzymes
Operon
When genes are arranged in a cluster on a chromosome and transcribed by a single promoter. Operons are common in bacteria but rare in eukaryotes
Operator
The sequence within the promoter region that a repressor protein binds to. The operator is a cis-regulatory sequence/genetic switch
Tryptophan repressor mechanism
Low tryptophan concentration- RNA polymerase binds to the promoter and transcribes the 5 genes in the tryptophan operon. High tryptophan concentration- the repressor protein is activated. It binds to the operator and blocks the binding of RNA polymerase to the promoter. When the tryptophan concentration drops, the repressor falls off of the DNA and allows the polymerase to transcribe the operon
Catabolite activator protein (CAP)
Activates transcription of genes that enable E. coli to utilize carbon sources other than glucose. It is activated when glucose levels are low
Catabolite activator protein (CAP) mechanism
When glucose levels fall, cyclic AMP (cAMP) levels increase. cAMP binds to CAP and allows it to bind sequences near the promoter, activating transcription
Lac operon
An E. coli operon that codes for proteins that transport and break down lactose. CAP activates these genes when glucose levels decrease, and CAP must be bound for the operon to be activated. However, lactose must also be present for the operon to be activated
Lac repressor
The lac repressor shuts off the operon in the absence of lactose. Therefore, the repressor is activated in low lactose conditions
Lac repressor mechanism (3)
- Low levels of β-galactosidase convert lactose to allolactose
- Allolactose binds lac repressor & releases it from operator
- lac genes transcribed
Which lac genes are transcribed by the lac repressor? (3)
- lacZ: β-galactosidase- cleaves lactose to glucose/galactose
- lacY: permease- Symporter - uses energy from H+ gradient for lactose uptake
- lacA: thiogalactoside transacetylase - no clear role
Lactose structure
Contains a beta 1-4 glycosidic bond- an O bond is between the 4th carbon on one ring and the 1st carbon on the other
Allolactose structure
Contains a beta 1-6 glycosidic bond
DNA looping
Genetic switches are generally located close to the start point of transcription, but this isn’t always true. If the switches are located farther away, the intervening DNA is looped out, allowing a protein bound at a distant site on the DNA to contact RNA polymerase. The DNA acts as a tether so the proteins will collide. Looping occurs in the regulation of almost every eukaryotic gene
The lac operon is only expressed when
Glucose is absent and lactose is present
Transcription factors in bacteria
The only TF that bacteria have is sigma factor, compared to the many TFs found in eukaryotes
Transcriptional regulators
A type of eukaryotic transcription factor that promotes activation or repression
Mediator
A eukaryotic intermediary between regulatory proteins and RNA polymerase
Are there operons in eukaryotic cells?
No, each gene is regulated individually
Gene control region
Eukaryotes- Contains the promoter and many regulatory sequences
Regulatory sequences
Eukaryotes- Sites where transcription factors bind and control the rate of assembly at the promoter. Regulatory sequences may be spread over vast sequences
3 methods of transcriptional activation in eukaryotes
- Direct interaction of regulatory TF w/ 1 or more general TF
- Interaction of regulatory TF with Mediator, where it facilitates assembly of RNA Pol and general TFs at promoter
- Modification of chromatin structure around promoter
What is always the result of transcriptional activation in eukaryotes?
Attract, position, & modify general TFs, Mediator, and Pol II at promoter
Function of eukaryotic transcription regulators
The regulators usually resemble in groups at their genetic switches. Multisubunit proteins called coactivators and co-repressors can assemble with them. An individual transcription regulator can participate in more than one regulatory complex. The ultimate function of the complex depends on its components
Eukaryotic assembly of RNA polymerase
Genetic switches enhance the rate of transcription initiation. Assemblies of activator proteins both attract and position RNA polymerase 2 at the promoter, and release it so that transcription can begin. Some activator proteins bind directly to general transcription factors and accelerate their assembly on a promoter, others attract coactivators that perform the biochemical tasks that initiate transcription
Modification of chromatin structure
Regulatory transcription factors recruit histone modifying enzymes, ATP-dependent chromatin remodeling complexes, and histone chaperones, which relax chromatin. The alterations in chromatin structure provide greater access to DNA and facilitate the assembly of general transcription factors at the promoter
Steps in chromatin remodeling (4)
- Nucleosome sliding allows access of transcription machinery to DNA
- Transcription machinery assembles on nucleosome-free DNA
- Histone variants allow greater access to nucleosomal DNA
- Specific patterns of histone modification destabilize compact forms of chromatin and attract components of transcription machinery
Effect of Histone acetylation-deacetylation
Chromatin is relaxed following acetylation
Steps of chromatin relaxation (4)
- A transcription activator binds to DNA packaged into chromatin and attracts a histone acetyl transferase
- The transferase acetylates two lysines on the histones H3 and H4
- A histone kinase, which was also attracted by the transcription activator, phosphorylates histone H3
- The general transcription factor TFIID and a chromatin remodeling complex bind to the chromatin to promote transcription initiation
How do transcription factors recognize acetylated histones?
TFIID and the remodeling complex recognize acetylated histone tails through a bromodomain, found in a subunit of each protein complex
Synergistic transcription
When several factors work together to enhance the reaction rate, they increase the rate more than the combination of either factor
Role of eukaryotic repressors
Repressors can both depress the rate of transcription below the default value and shut off genes that were previously activated, Repressors can hinder activators or disruption the interaction with a general transcription factor or mediator
Mechanisms of eukaryotic transcriptional repressors (3)
- Recruitment of chromatin remodeling complexes
- Recruitment of histone deacetylases
- Recruitment of histone methyl transferases
Riboswitches
Short sequences of RNA that change their conformation when they bind small molecules, like metabolites. Each riboswitch is specific to a small molecule, and conformational changes are used to regulate gene transcription. They are located near the 5’ end of mRNA, and fold when it’s synthesized, blocking (or allowing) RNA polymerase. They are very common in bacteria
Guanine riboswitch
There are riboswitches for purine (adenine and guanine) biosynthetic genes in bacteria. When guanine levels are low, the riboswitch allows transcription of genes. When guanine levels are high, the riboswitch binds and undergoes a conformational change. This causes early termination of transcription when the mRNA transcript is pulled out, like an RNA hairpin
How do B cells change their antibody structure as they develop?
During the development of B cells, there is a switch from the synthesis of membrane bound to secreted antibody molecules. Early on, the B cell antibody is anchored in the plasma membrane, but when the B cell is stimulated by antigen, it multiples and begins secreting its antibody. The secreted form of the antibody is identical to the membrane-bound form, except for at the C-terminus. At this point, the membrane bound form has a string of hydrophobic amino acids that goes through the plasma membrane. The secreted form has a shorter string of hydrophilic amino acid
B cell polyadenylation
The switch in antibody structure is generated through a change in the site of RNA cleavage and polyadenylation. An increase in the concentration of cleavage stimulation factor (CSTF) promotes RNA cleavage. The first cleavage/poly-A addition site that a transcribing RNA polymerase encounters is suboptimal and is usually skipped in unstimulated B lymphocytes. This leads to the production of a longer RNA transcript. When activated to produce antibodies, B cells increase their CSTF concentration, cleavage occurs at the suboptimal site, and a shorter transcript is produced
RNA interference (RNAi)
Short single-stranded RNAs (20-30 nucleotides) serve as guide RNAs that reorganize and bind other RNAs in the cell. This is an evolutionarily ancient defense mechanism that is triggered by the presence of double stranded RNA. Viral RNA is degraded as in miRNA
microRNAs (miRNA)
Pair with specific miRNAs and fine-tune their stability and translation. The miRNA precursors are synthesized by RNA polymerase 2 and are polyadenylated. Then, they are assembled with proteins to form an RNA-induced silencing complex (RISC). The RISC seeks out target mRNAs by searching for complementary nucleotide sequences.
RNA-induced silencing complex (RISC)
RISC searches for complementary mRNAs. The search is facilitated by the Argonaute protein, which is part of the RISC. It holds the 5’ region of the miRNA so that it is optimally positioned for base-pairing to another mRNA molecule. Usually, at least 7 nucleotides base pair
Regulation by miRNA in plants (3)
- miRNA directs RISC to mRNA via base pairing
- Argonaute cleaves mRNA
- Removal of poly-A tails, degradation by exonucleases
Regulation by miRNA in animals
Translation is repressed/poly-A tail is shortened, and capping/degradation in processing bodies (P-bodies)
Dicer nuclease
Cleaves dsRNA into small interfering RNA (siRNA) (~ 23 bps)
Argonaute
Makes siRNA single stranded
RNA interference and heterochromatin formation (3 steps)
RNAi can silence transcription through heterochromatin formation and histone methylation.
1. siRNA assembles w/ Argonaute & other proteins to form RNA-induced transcriptional silencing (RITS) complex
2. siRNA guides RITS to complementary RNA emerging from Pol II
3. RITS recruits histone modifying & chromatin remodeling proteins
Use of RNAi in the lab
When RNA is degraded by RNAi, those degraded RNA fragments are used to make more siRNAs, and the cycle continues. Short RNA fragments can be passed on to progeny
siRNA
Found in plants and lower animals, maybe mammals. Composed of double stranded linear RNA. Around 23 base pairs in length and has specific complementarity. Function- gene silencing in organisms lacking complex immune systems
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) type 2 system (5 steps)
A naturally occurring system in bacteria that combats bacteriophage infection.
1. When a phage infects bacteria, pieces of phage DNA are integrated into the crispr locus. 2. Then, the crispr locus is transcribed and small guide RNA (sgRNA) is formed.
3. Transcripts for Cas nuclease are formed
4. Cas is translated, sgRNA is assembled onto Cas
5. sgRNA guides Cas nuclease to complementary phage DNA, and phage DNA is cut
