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Learning objectives associated with Winter_2021_Bis2A_Facciotti_Reading_24
- Given a metabolic pathway, propose and justify potential molecules (such as positive/negative regulators, transcription factors, etc.) to regulate key steps in the pathway.
- Given some specific constraints on a regulatory circuit, predict the behavior of the circuit under the possible influence of environmental or genetic perturbations (changes).
- Be able to rank the different levels of regulation discussed in class by energy efficiency for the cell and create statements that illustrate the logic used in the rankings.
- Using a regulatory network, accurately interpret relationships between genes/proteins in the network and predict potential targets of regulation by various products in the pathway.
Examples of Bacterial Gene Regulation
This section describes two examples of transcriptional regulation in bacteria. Be on the lookout in class, in discussion, and in the study-guides for extensions of these ideas and use these to explain the regulatory mechanisms used for regulating other genes.
Gene Regulation Examples in E. coli
The Role of the Promoter
The first level of control of gene expression is at the promoter itself. Some promoters recruit RNA polymerase and turn those DNA-protein binding events into transcripts more efficiently than other promoters. This intrinsic property of a promoter, it's ability to produce transcript at a particular rate,
UC Davis Undergraduate Connection:
A group of UC Davis students interested in synthetic biology used this idea to create synthetic
Example #1: Trp Operon
Logic for regulating tryptophan biosynthesis
E. coli, like all organisms, needs to either synthesize or consume amino acids to survive. The amino acid tryptophan is one such amino acid. E. coli can either import tryptophan from the environment (eating what it can scavenge from the world around it) or synthesize tryptophan de novo using enzymes encoded by five genes. These five genes
Organization of the trp operon
The five genes encoding tryptophan biosynthesis enzymes
sequentially on the chromosome and are under the control of a single promoter - i.e. natural selection
into an operon. Just before the coding region is the transcriptional start site. This is, as the name implies, the location where the RNA polymerase starts a new transcript. The promoter sequence is further upstream of the transcriptional start site.
A DNA sequence called an "operator"
between the promoter and the first
A few more details regarding TF binding sites
We should note that the use of the term "operator" is limited to just a few regulatory systems and almost always refers to the binding site for a negatively acting transcription factor. Conceptually what you need to remember is that there are sites on the DNA that interact with regulatory proteins, allowing them to perform their appropriate function (e.g. repress or activate transcription). This theme will repeat universally across biology whether the "operator" term
While the specific examples you will be show depict TF binding sites in their known locations, these locations are not universal to all systems. Transcription factor binding sites can vary in location relative to the promoter. There are some patterns (e.g. positive regulators are often upstream of the promoter and negative regulators bind downstream), but these generalizations are not true for all cases. Again, the key thing to remember is that transcription factors (both positive and negatively acting) have binding sites with which they interact to help regulate the initiation of transcription by RNA polymerase.
The five genes that required to synthesize tryptophan in E. coli group next to each other in the
Regulation of the trp operon
When tryptophan is present in the cell: two tryptophan molecules bind to the
and translated, and tryptophan
Since the transcription factor actively binds to the operator to keep the genes turned off, the
" and the proteins that bind to the operator to silence trp expression are negative regulators.
Possible NB Discussion Point
Suppose nature took a different approach to regulating the trp operon. Propose a method for regulating the expression of the
Watch this video to learn more about the
Example #2: The lac operon
Rationale for studying the lac operon
In this example, we examine the regulation of genes encoding proteins whose physiological role is to import and assimilate the disaccharide lactose, the lac operon. The story of regulating lac operon is a common example used in many introductory biology classes to illustrate basic principles of inducible gene regulation. We describe this example second because it is, in our estimation, more complicated than the previous example involving the activity of a single negatively acting transcription factor.
regulation of the lac operon is a wonderful example of how the coordinated activity of both positive and negative regulators around the same promoter can integrate multiple different sources of cellular information to regulate the expression of genes.
As you go through this example, keep in mind the last point. For many Bis2a instructors, it is more important for you to learn the lac operon story and guiding principles than it is for you to memorize the logic table presented below. When this is the case, the instructor will usually let you know. These instructors often deliberately do NOT include exam questions about the lac operon. Rather, they will test you on whether you understood the fundamental principles underlying the regulatory mechanisms that you study using the lac operon example. If it's not clear what the instructor wants, ask.
The utilization of lactose
Lactose is a disaccharide composed of the hexoses glucose and galactose. We commonly encounter lactose in milk and some milk products. As one can imagine, the disaccharide can be an important food-stuff for microbes that can use its two hexoses. coli can use multiple different sugars as energy and carbon sources, including
and the lac operon is a structure that encodes the genes necessary to
and process lactose from the local environment. coli, however, does not frequently encounter lactose, and therefore the genes of the lac operon must typically
(i.e. "turned off") when lactose is absent. Driving transcription of these genes when lactose is absent would waste precious cellular energy.
lactose is present, it would make logical sense for the genes responsible for using the sugar to
(i.e. "turned on"). So far, the story is very similar to that of the tryptophan operon described above.
However, there is a catch. Experiments conducted in the
by Jacob and Monod showed that E. coli prefers to use all the glucose present in the environment before it uses lactose. This means that the mechanism used to decide
to express the lactose utilization genes must be able to integrate two types of information (1) the concentration of glucose and (2) the concentration of lactose. While this could theoretically
in multiple ways, we will examine how the lac operon accomplishes this by using multiple transcription factors.
The transcriptional regulators of the lac operon
The lac repressor - a direct sensor of lactose
As noted, the lac operon normally has very low to no transcriptional output in the absence of lactose. This is because of two factors: (1) the constitutive promoter strength for the operon is relatively low and (2) the constant presence of the LacI repressor protein negatively influences transcription. This protein binds to the operator site near the promoter and blocks RNA polymerase from transcribing the lac operon genes.
CAP protein - an indirect sensor of glucose
In E. coli, when glucose levels drop, the small molecule cyclic AMP (
is a common signaling molecule that
in glucose and energy metabolism in many organisms. When glucose levels decline in the cell, the increasing concentrations of
allow this compound to bind to the positive transcriptional regulator called catabolite activator protein (CAP) - also referred to as CRP.
-CAP complex has many sites throughout the E. coli genome and many of these sites
near the promoters of many operons that control the processing of various sugars.
In the lac operon, the
-CAP binding site
upstream of the promoter. Binding of
-CAP to the DNA helps to recruit and keep RNA polymerase to the promoter. The increased occupancy of RNA polymerase to its promoter
results in increased transcriptional output. Here, the CAP protein is acting as a positive regulator.
Note that the CAP-
complex can, in other operons, also act as a negative regulator depending upon where the binding site for CAP-
relative to the RNA polymerase binding site.
Putting it all together: Inducing expression of the lac operon
For the lac operon to
A more nuanced view of lac repressor function
The description of the lac repressor's function correctly describes the logic of the control mechanism used around the
The lac operon regulatory region depicting the promoter, three lac operators, and CAP binding site.
The lac repressor tetramer (blue) depicted binding two operators on a strand of looped DNA (orange).
Eukaryotic Gene Regulation
As previously noted, regulation is all about decision making. Gene regulation, as a general topic, relates to
about the functional expression of genetic material. Whether the final product is an RNA species or a protein, the production of the final expressed product requires processes that take multiple steps. We have spent some time discussing some of these steps (i.e. transcription and translation) and some mechanisms that nature uses for sensing cellular and environmental information to regulate the initiation of transcription.
When we discussed the concept of strong and weak promoters, we introduced the idea that regulating the amount (number of molecules) of a transcript produced from a promoter in some unit of time might also be important for function. This should not be entirely surprising. For a protein-coding gene, the more transcript produced, the greater potential there is to make more protein. This might be important when making a lot of a particular enzyme is key for survival. In other cases, the cell needs only a little of a specific protein and making too much would be a waste of cellular resources.
, the cell may prefer low levels of transcription. Promoters of differing strengths can accommodate these varying needs. Regarding transcript number, we also briefly mentioned that synthesis is not the only way to regulate abundance. Degradation processes are also important to consider.
In this section, we add to these themes by focusing on eukaryotic regulatory processes. Specifically, we examine - and sometimes re-examine - the multiple steps required to express genetic material in eukaryotic organisms in
regulation. We want you not only to think about the processes but also to recognize that each step in the process of expression is also an opportunity to fine tune not only the abundance of a transcript or protein but also its functional state, form (or variant), and/or stability. Each of these additional factors may also be vitally important to consider for influencing the abundance of conditionally specific functional variants.
Structural differences between bacterial and eukaryotic cells influencing gene regulation
The defining hallmark of the eukaryotic cell is the nucleus, a double membrane that encloses the cell's hereditary material. In order to efficiently fits the organism's DNA into the confined space of the nucleus, the DNA is first packaged and organized by protein into a structure called chromatin. This packaging of the nuclear material reduces access to specific parts of the chromatin. Some elements of the DNA are so tightly packed that the transcriptional machinery cannot access regulatory sites like promoters. This means that one of the first sites of transcriptional regulation in eukaryotes must be the control access to the DNA itself. Chromatin proteins can be subject to enzymatic modification that can influence whether they bind tightly (limited transcriptional access) or more loosely (greater transcriptional access) to a segment of DNA
This process of modification - whichever direction
first - is reversible. Therefore, DNA can
and made available when the "time is right".
The regulation of gene expression in eukaryotes also involves
same additional fundamental mechanisms discussed in the module on bacterial regulation (i.e. the use of strong or weak promoters, transcription factors, terminators etc.) but the actual number of proteins involved is typically much greater in eukaryotes than bacteria or archaea.
The post-transcriptional enzymatic processing of RNA that occurs in the nucleus and the export of the mature
to the cytosol are two additional difference between bacterial and eukaryotic gene regulation. We will consider this level of regulation in more detail below.
Depiction of some key differences between the processes of bacterial and eukaryotic gene expression. Note in this case
DNA Packing and Epigenetic Markers
The DNA in eukaryotic cells
The first level of organization, or packing, is the winding of DNA strands around histone proteins. Histones package and order DNA into structural units called nucleosomes, which can control the access of proteins to specific DNA regions. Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string. These beads (nucleosome complexes) can move along the string (DNA) to alter which areas of the DNA are accessible to transcriptional machinery. While nucleosomes can move to open the chromosome structure to expose a segment of DNA, they do so in a very controlled manner.
DNA folds around histone proteins to create (a) nucleosome complexes. These nucleosomes control the access of proteins to the underlying DNA. When viewed through an electron microscope (
How the histone proteins move depends on chemical signals found on both the histone proteins and on the DNA. These chemical signals are chemical tags added to histone proteins and the DNA that tell the histones if a chromosomal region should be "open" or "closed". The figure below depicts modifications to histone proteins and DNA. These tags are not permanent, but may
Nucleosomes can slide along DNA. When nucleosomes
Possible NB Discussion Point
In the later maturation phase of sperm cells, histones (containing high numbers of lysine amino acids) are replaced by protamines, which are small, nuclear proteins that are very rich in arginine amino acids. This process is said to be essential for sperm head condensation and DNA stabilization. Based on this information, what comparisons can you draw between protamines and histones? Why is it significant that there are high numbers of lysine and arginine in histones and protamines? For what reasons do you think protamines replace histones in sperm but not other cells?
Epigenetic changes do not result in permanent changes in the DNA sequence. Epigenetic changes alter the chromatin structure (protein-DNA complex) to allow or deny access to transcribe genes. DNA modification such as methylation on cytosine nucleotides can either recruit repressor proteins that block RNA polymerase's access to transcribe a
Regulation of gene expression through chromatin remodeling
View this video that describes how epigenetic regulation controls gene expression.
Eukaryotic gene structure and RNA processing
Eukaryotic gene structure
Many eukaryotic genes, particularly those encoding protein products,
The parts of a typical discontinuous eukaryotic gene. Attribution:
Parts of a generic eukaryotic gene include familiar elements like a promoter and terminator. Between those two elements, the region encoding all the elements of the gene that have the potential to
(they have no stop codons), like in bacterial systems,
the open reading frame (ORF). Enhancer and/or
elements are regions of the DNA that recruit regulatory proteins. These can be relatively close to the promoter, like in bacterial systems, or thousands of nucleotides away. Also present in many bacterial transcripts, 5' and 3' untranslated regions (UTRs) also exist. These regions of the gene encode segments of the
which, as their names imply,
and sit 5' and 3', respectively, to the ORF. The UTRs typically encode some regulatory elements critical for regulating transcription or steps of gene expression that occur post-transcriptionally.
The RNA species resulting from the transcription of these genes are also discontinuous and must therefore
before exiting the nucleus to
or used in the cytosol as mature RNAs. In eukaryotic systems this includes RNA splicing, 5' capping, 3' end cleavage and polyadenylation. This series of steps is a complex molecular process that must occur within the closed confines of the nucleus. Each one of these steps provides an opportunity for regulating the abundance of exported transcripts and the functional forms that these transcripts will take. While these would be topics for more advanced courses, think about how to frame
following topics as subproblems of the Design Challenge of genetic regulation. If nothing else,
appreciate the highly orchestrated molecular dance that must occur to express a gene and how this is a stunning bit of evolutionary engineering.
Like in bacterial systems, eukaryotic systems must assemble a pre-initiation complex at and around the promoter sequence to start transcription. The complexes that assemble in eukaryotes serve many of the same function as those in bacterial systems, but they are significantly more complex, involving many more regulatory proteins. This added complexity allows for greater regulation and for the assembly of proteins with functions that occur predominantly in eukaryotic systems. One of these additional functions is the "capping" of nascent transcripts.
In eukaryotic protein-coding genes, the RNA that is first produced
. The "pre" prefix signifies that this is not the full mature
and that it first requires some processing. The modification known as 5'-capping occurs after the pre-
is about 20-30 nucleotides long. At this point the pre-RNA typically receives its first post-transcriptional modification, a 5'-cap. The "cap" is a chemical modification - a 7-
- whose addition to the 5' end of the transcript
by multiple enzymes called the capping enzyme complex (CEC) a group of multiple enzymes that carry out sequential steps involved in adding the 5'-cap. The CEC binds to the RNA polymerase very early in transcription and carries out a modification of the 5' triphosphate, the subsequent transfer of at GTP
(connecting the two nucleotides using a unique 5'-to-5' linkage), the methylation of the newly transferred guanine, and in some transcripts the additional modifications to the first few nucleotides. This 5'-cap appears to function by protecting the emerging transcript from degradation and
by RNA binding proteins known as the cap-binding complex (CBC). There is some evidence that this modification and the proteins bound to it play a role in targeting the transcript for export from the nucleus. Protecting the nascent RNA from degradation is not only important for conserving the energy invested in creating the
in regulating the abundance of
role of the 5'-cap in guiding the transcript for export will directly help to regulate not only the amount of transcript that
the amount of transcript that
to the cytoplasm that has the potential to
The structure of a typical 7-
Cells must process nascent transcripts into mature RNAs by joining exons and removing the intervening introns. They
complex of RNA and proteins called the spliceosome. The spliceosome complex assembles on the nascent transcript and most times the decisions about which introns to combine into a mature transcript
at this point. How these decisions
but involves the recognition of specific DNA sequences at the splice sites by RNA and protein species and several catalytic events. It is interesting to note that the catalytic portion of the spliceosome
of RNA rather than protein. Recall that the ribosome is another example of
-protein complex where the RNA serves as the primary catalytic component. The selection of which splice variant to make is a form of regulating gene expression.
rather than influencing abundance of a transcript, alternative splicing allows the cell to decide about which
is common in eukaryotic systems and
to be important in different stages of development in multicellular organisms and in defining the functions of different cell types.
gene products from a single gene whose transcription initiation
from a single transcriptional regulatory site (by
of which end-product to produce post-transcriptionally) obviates the need to create and maintain independent copies of each gene in different parts of the genome and evolving independent regulatory sites. Therefore, the ability to form multiple
from a single coding region is though to be evolutionarily
because it enables some efficiency in DNA coding, minimizes transcriptional regulatory complexity, and may lower the energy burden of maintaining more DNA and protecting it from mutation. Some examples of
outcomes of alternative splicing can include: the generation of enzyme variants with differential substrate affinity or catalytic rates;
; entirely new functions, via the swapping of protein domains can
. These are just a few examples.
One additional interesting outcome of alternative splicing is the introduction of stop codons that can, through a mechanism that seems to require translation, lead to the targeted decay of the transcript. This means that, besides the control of transcription initiation and 5'-capping, we can also consider alternative splicing as one of the regulatory mechanisms that may influence transcript abundance. The effects of alternative splicing are therefore potentially broad - from complete loss of function to novel and diversified function to regulatory effects.
A figure depicting some different modes of alternative splicing illustrating how different splice variants can lead to different protein forms.
3' end cleavage and polyadenylation
RNA Stability andmicroRNAs
Besides the modifications of the pre-RNA described above and the associated proteins that bind to the nascent and transcripts, there are other factors that can influence the stability of the RNA in the cell. One example are elements called
Fully processed, mature
We know many additional details of the processes described above to some level of detail, but many more questions remain to
. For the sake of
to form a model of the steps that occur in the production of a mature transcript in eukaryotic organisms. We have painted a picture with very broad strokes, trying to present a scene that reflect what happens
in all eukaryotes. Besides learning the key differentiating features of eukaryotic gene regulation, we would also like for Bis2a students to think of each of these steps as an opportunity for Nature to regulate gene expression
and to rationalize how deficiencies or changes in these pathways - potentially introduced through mutation - might influence gene expression.
While we did not explicitly bring up the Design Challenge or Energy Story
these formalisms are equally adept at helping you to make some sense of what is being described. We encourage you to try making an Energy Story for various processes. We also encourage you to use the Design Challenge rubric to reexamine the stories above: identify problems that need solving; hypothesize potential solutions and criteria for success. Use there formalisms to dig deeper and ask new questions/identify new problems or things that you don't know about the processes is what experts do. Chances are that doing this suggested exercise will lead you to identify a direction of research that someone has already pursued (you'll feel
smart about that!). Alternatively, you may raise some brand new question that no one has thought of yet.
Control of Protein Abundance
The initiation complex and translation rate
An increase in phosphorylation levels of
Chemical Modifications, Protein Activity, and Longevity
by nucleic acids, proteins can also
with the addition of groups including methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins can regulate their activity or the
time they exist in the cell. Sometimes these modifications can regulate where a protein
in the cell—for example, in the nucleus, the cytoplasm, or attached to the plasma membrane.
Chemical modifications can occur in response to external stimuli such as stress, the lack of nutrients, heat, or ultraviolet light exposure.
regulating the function of the proteins themselves, if these changes occur on specific proteins they can alter epigenetic accessibility (
histone modification), transcription (transcription factors),
stability (RNA binding proteins), or translation (
-2) thus feeding back and regulating various parts of the process of gene expression.
modification to regulatory proteins, this can be an efficient way for the cell
the levels of specific proteins in response to the environment by regulating various steps
The addition of an ubiquitin group has another function - it marks that protein for degradation.
is a small molecule that acts like a flag
that the tagged proteins should
to an organelle called the proteasome. This organelle is a large multi-protein complex that functions to cleave proteins into smaller pieces that can then
(the addition of
tag), therefore helps to control gene expression by altering the functional lifetime of the protein product.
In conclusion, we see that gene regulation is complex and that