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2020_Spring_Bis2a_Facciotti_Lecture_25 - Biology

2020_Spring_Bis2a_Facciotti_Lecture_25 - Biology


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Learning Objectives Associated with 2020_Spring_Bis2a_Facciotti_Lecture_25

  • Given the central dogma, propose a rationale for the need to regulate each step, including biomolecule degradation.
  • Given information regarding the allosteric regulation of a DNA binding protein, predict what effect changing the concentration of the allosteric regulator would have on the binding of a transcription factor to a regulatory element.
  • Describe the roles of both positive and negative transcriptional regulators in the control of gene expression.
  • Draw models that help explain how the allosteric binding of small molecules to both positively and negatively acting transcription factors can in both cases explain their ability to either “turn up” or “turn down” transcription in a small-molecule concentration-dependent manner.

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 DNA of bacteria andarchaeaare usually organizedinto one or more circular chromosomes in the cytoplasm. The dense aggregate of DNA that canbe seenin electron micrographsis calledthe nucleoid. In bacteria and archaea,genes,whose expression needs tobe tightly coordinated(e.g. genes encoding proteins involved in the same biochemical pathway)are often groupedclosely together in the genome. When the expression of multiple genesis controlledby the same promoter and a single transcriptis producedthese expression unitsare calledoperons. For example, in the bacterium Escherichia coli all the genes needed to utilize lactoseare encodednext to one another in the genome. We call this arrangement the lactose (or lac) operon. In many bacteria and archaea nearly 50% of all genesare encodedinto operons of two or more genes.

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,is referredto aspromoterstrength. The stronger the promoter,the more RNA is madein any given time period. Promoter strength canbe "tuned" by Nature invery smallor very large steps by changing the nucleotide sequence the promoter (e.g. mutating the promoter). This results in families of promoters with different strengths that canbe used tocontrol the maximum rate of gene expression for certain genes.

UC Davis Undergraduate Connection:

A group of UC Davis students interested in synthetic biology used this idea to create syntheticpromoterlibraries for engineering microbes as part of their design project for the 2011 iGEM competition.

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 genesresidenext to each other in the E. coli genome in what we call the tryptophan (trp) operon (Figure below). If tryptophan is present in the environment, then E. coli need not synthesize it and the switch controlling the activation of the genes in the trp operon switches off. However, when environmental tryptophan availability is low, the switch controlling the operonis turnedon,transcription is initiated, the genesare expressed, and the organism synthesizes tryptophan. See the figure and paragraphs below for a mechanistic explanation.

Organization of the trp operon

The five genes encoding tryptophan biosynthesis enzymes

are arranged

sequentially on the chromosome and are under the control of a single promoter - i.e. natural selection

organized thesegenes

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"

is also encoded

between the promoter and the first trp coding gene. This operator is the DNA sequence to which the transcription factor protein will bind.

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

is used

.

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 thetrpoperon. When tryptophan is plentiful, two tryptophan molecules bind to the transcription factor and allow the TF-tryptophan complex to bind at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes. When tryptophan is absent, the transcription factor does not bind to the operator andthe genes are transcribed.
Attribution:Marc T. Facciotti (own work)

Regulation of the trp operon

When tryptophan is present in the cell: two tryptophan molecules bind to the trp repressor protein. When tryptophan binds to the transcription factor it causes a conformational change in the protein which now allows the TF-tryptophan complex to bind to the trp operator sequence. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding and transcribing the downstream genes. When tryptophan is not present in the cell, the transcription factor does not bind to the operator; therefore, the transcription proceeds, the tryptophan utilization genes

are transcribed

and translated, and tryptophan

is thus synthesized

.

Since the transcription factor actively binds to the operator to keep the genes turned off, the trp operon

is said

to

be "negatively regulated

" 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 trp operon with a positive regulator instead of a negative regulator. Describe how this might work. (Hint: we ask this kind of question on exams)


External link

Watch this video to learn more about the trp operon.

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.

By contrast, the

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

lactose

and the lac operon is a structure that encodes the genes necessary to

acquire

and process lactose from the local environment. coli, however, does not frequently encounter lactose, and therefore the genes of the lac operon must typically

be repressed

(i.e. "turned off") when lactose is absent. Driving transcription of these genes when lactose is absent would waste precious cellular energy.

By contrast, when

lactose is present, it would make logical sense for the genes responsible for using the sugar to

be expressed

(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

1950's

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

whether or not

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

be accomplished

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.By contrast, iflactose is present, lactose will bind to the LacI protein, inducing a conformational change that prevents LacI-lactose complex from binding to its binding sites. Therefore, when lactose is present, the negative regulatory LacIis not boundto its binding site and transcription of lactose using genes can proceed.

CAP protein - an indirect sensor of glucose

In E. coli, when glucose levels drop, the small molecule cyclic AMP (cAMP) accumulates in the cell.

cAMP

is a common signaling molecule that

is involved

in glucose and energy metabolism in many organisms. When glucose levels decline in the cell, the increasing concentrations of

cAMP

allow this compound to bind to the positive transcriptional regulator called catabolite activator protein (CAP) - also referred to as CRP.

cAMP

-CAP complex has many sites throughout the E. coli genome and many of these sites

are located

near the promoters of many operons that control the processing of various sugars.

In the lac operon, the

cAMP

-CAP binding site

is located

upstream of the promoter. Binding of

cAMP

-CAP to the DNA helps to recruit and keep RNA polymerase to the promoter. The increased occupancy of RNA polymerase to its promoter

, in turn,

results in increased transcriptional output. Here, the CAP protein is acting as a positive regulator.

Note that the CAP-

cAMP

complex can, in other operons, also act as a negative regulator depending upon where the binding site for CAP-

cAMP

complex

is located

relative to the RNA polymerase binding site.

Putting it all together: Inducing expression of the lac operon

For the lac operon tobe activated,two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present, willthelacoperonbe transcribed. When this conditionis achievedtheLacI-lactose complex dissociates the negative regulator from near the promoter, freeing the RNA polymerase to transcribe the operon's genes. HighcAMP(indirectly indicative of low glucose) levels trigger the formation of the CAP-cAMPcomplex. This TF-inducer pair now bind near the promoter and actto positively recruitthe RNA polymerase. This added positive influence boosts transcriptional outputandlactose canbe efficiently utilized.The mechanistic output of other combinations of binary glucose and lactose conditions are describedin the table below and in the figure that follows.

Truth Table for Lac Operon

Transcription of the lac operonis carefully regulatedso that its expression only occurs when glucoseis limitedand lactose is present to serve as an alternative fuel source.
Attribution:Marc T. Facciotti (own work)

Signals that Induce or Repress Transcription of the lac Operon
GlucoseCAP bindsLactoseRepressor bindsTranscription
+--+No
+-+-Some
-+-+No
-++-Yes

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 thelacpromoter. However, the molecular description of binding sites is a bit overly simplified. In reality, the lac repressor has three similar, but not identical, binding sites called Operator 1, Operator 2, and Operator 3. Operator 1 is very close to the transcript start site (denoted +1).Operator 2 is locatedabout +400nt into the coding region of theLacZprotein.Operator 3 is locatedabout -80nt before the transcript start site (just "outside" of the CAP binding site).

The lac operon regulatory region depicting the promoter, three lac operators, and CAP binding site.The coding region for the Lac Z protein is also shownrelative to the operator sequences. Note that two of the operators are in the protein coding region - there are multiple differenttypes ofinformation simultaneously encoded in the DNA.
Attribution:Marc T. Facciotti (own work)

The lac repressor tetramer (blue) depicted binding two operators on a strand of looped DNA (orange).
Attribution:Marc T. Facciotti (own work) - Adapted from Goodsell (https://pdb101.rcsb.org/motm/39)

Eukaryotic Gene Regulation

Regulation overview

As previously noted, regulation is all about decision making. Gene regulation, as a general topic, relates to

deciding

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.

Here

, 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

the context of

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

is considered

first - is reversible. Therefore, DNA can

be dynamically sequestered

and made available when the "time is right".

The regulation of gene expression in eukaryotes also involves

some of the

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

mRNA

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 casethe presence ofhistone and histone modifiers, the splicing of pre-mRNA, and the export of the mature RNA from the nucleus as key differentiators between the bacterial and eukaryotic systems.
Attribution:Marc T. Facciotti (own work)

DNA Packing and Epigenetic Markers

The DNA in eukaryotic cellsis precisely wound, folded, and compacted into chromosomes so it will fit into the nucleus. The nucleusalso organizes the DNAso key proteinscan easily access specific segments of the chromosomesas needed. Areas of the chromosomes that are more tightly compacted will be harder for proteins to bind and therefore lead to reduced gene expression of genes encoded in those areas. Loosely compacted regions of the genome will be easier for proteins to access, thus increasing the likelihood thatagene will be transcribed. Discussed here are how cells regulate the density of DNA compaction.

DNA packing

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 (b), the nucleosomes look like beads on a string. (credit “micrograph”: modification of work by Chris Woodcock)

Histone Modification

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 maybe addedor removed as needed. They are chemical modifications (phosphate, methyl, or acetyl groups) that attach to specific amino acids in the histone proteins or to the nucleotides of the DNA. The tags do not alter the DNA base sequence, but theydoalter how tightly wound the DNA is around the histone proteins. DNA is a negatively charged molecule; therefore, changes in the histone's charge will change how tightly wound the DNA molecule will be. When unmodified, the histone proteins have a large positive charge; by adding chemical modifications like acetyl groups, the charge becomes less positive.

Nucleosomes can slide along DNA. When nucleosomesare spacedclosely together (top), transcription factors cannot bind andgene expression is turnedoff. When the nucleosomesare spacedfar apart (bottom),the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing.


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?


DNA Modification

The DNA molecule itself can also be modified. This occurs within very specific regions called CpG islands. These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) often found in the promoter regions of genes. When this configuration exists, the cytosine member of the pair can be methylated (a methyl group is added). This modification changes how the DNA interacts with proteins, including the histone proteins that control access to the region. Highly methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive.

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 ageneor they can aid in compacting the DNA to block all protein access to that area of the genome. These changes are reversible whereas mutations are not, however, epigenetic changes to the chromosome can alsobe inherited.
Source:modifiedfrom https://researcherblogski.wordpress....r/dudiwarsito/

Regulation of gene expression through chromatin remodelingis calledepigenetic regulation. Epigenetic means “around genetics.” The changes that occur to the histone proteins and DNA do not alter the nucleotide sequence and are not permanent. Instead, these changes are temporary (although they often persist through multiple rounds of cell division and canbe inherited) and alter the chromosomal structure (open or closed) as needed.

External link

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,are encodedon the genome discontinuously.Thecoding region is brokeninto pieces by intervening non-coding gene elements. We term the coding regions exons while the intervening non-coding elementsare termedintrons. The figure below depicts a generic eukaryotic gene.

The parts of a typical discontinuous eukaryotic gene. Attribution:Marc T. Facciotti (own work)

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

be translated

(they have no stop codons), like in bacterial systems,

is called

the open reading frame (ORF). Enhancer and/or

silencer

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

transcript,

which, as their names imply,

are not translated

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

be processed

before exiting the nucleus to

be translated

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

some of the

following topics as subproblems of the Design Challenge of genetic regulation. If nothing else,

begin to

appreciate the highly orchestrated molecular dance that must occur to express a gene and how this is a stunning bit of evolutionary engineering.

5' capping

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

is called

the pre-

mRNA

. The "pre" prefix signifies that this is not the full mature mRNA that will

be translated

and that it first requires some processing. The modification known as 5'-capping occurs after the pre-

mRNA

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-

methylguanosine

- whose addition to the 5' end of the transcript is enzymatically catalyzed 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

to this end

(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

is quickly bound

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

transcript

but

is clearly involved

in regulating the abundance of

fully-functional

transcript that

is produced

.

Moreover, the

role of the 5'-cap in guiding the transcript for export will directly help to regulate not only the amount of transcript that

is made

but, perhaps

more importantly,

the amount of transcript that

is exported

to the cytoplasm that has the potential to

be translated

.

The structure of a typical 7-methylguanylatecap. Attribution:Marc T. Facciotti (own work)

Transcript splicing

Cells must process nascent transcripts into mature RNAs by joining exons and removing the intervening introns. They

accomplish this

using a multicomponent 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

are made

at this point. How these decisions

are madeis still not completely understood

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

is made

of RNA rather than protein. Recall that the ribosome is another example of

a RNA

-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.

In this case

rather than influencing abundance of a transcript, alternative splicing allows the cell to decide about which

form of a transcript it makes

.

The alternative splice forms of genes that result in protein products of related structure but of varying function are known

as isoforms. The creation of isoforms is common in eukaryotic systems and

is known

to be important in different stages of development in multicellular organisms and in defining the functions of different cell types.

By

encoding multiple

possible

gene products from a single gene whose transcription initiation

is encoded

from a single transcriptional regulatory site (by

making the decision

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 isoforms from a single coding region is though to be evolutionarily

advantageous

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

possible

outcomes of alternative splicing can include: the generation of enzyme variants with differential substrate affinity or catalytic rates;

signal sequences that target proteins to various sub-cellular compartments can be changed

; entirely new functions, via the swapping of protein domains can

be created

. 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.
Attribution:Marc T. Facciotti (own work)

3' end cleavage and polyadenylation

One final modification is madeto nascent pre-mRNAsbefore they leave the nucleus - the cleavage of the 3' end and its polyadenylation.This two step process is catalyzedby two different enzymes (as depicted below) and may decorate the 3' end of transcripts with up to nearly 200 nucleotides. This modification enhances the stability of the transcript.Generally, themoreAsin the polyA tag the longer lifetime that transcript has. The polyA tag also seems to play a role in theexport of the transcriptfrom the nucleus. Therefore, the 3' polyA tag plays a role in gene expression by regulating functional transcript abundance andhow much is exportedfrom the nucleus for translation.

A two step process is involvedinmodifyingthe 3' ends of transcripts prior to nuclear exports. These include cutting transcripts just downstream of a conserved sequence (AAUAAA) and transferring adenylate groups. Both processes are enzymatically catalyzed.
Attribution:Marc T. Facciotti (own work)

MicroRNAs

RNA Stability and microRNAs

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 microRNAs. The microRNAs, or miRNAs, are short RNA molecules that are only 21–24 nucleotidesin length. The miRNAs are transcribed in the nucleus as longer pre-miRNAs. These pre-miRNAsare subsequently choppedinto mature miRNAs by a protein called dicer. These mature miRNAs recognize a specific sequence of a target RNA through complementary base pairing. miRNAs, however, also associate with a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). RISC binds a target mRNA, along with the miRNA, to degrade the target mRNA. Together, miRNAs and the RISC complex rapidly destroy the RNA molecule. As one might expect, the transcription of pre-miRNAsand their subsequent processingis also tightly regulated.

Nuclear export

Nuclear export

Fully processed, maturetranscripts,mustbe exportedthrough the nucleus.Not surprisingly thisprocess involves the coordination of a mature RNA species to whichare boundmany accessory proteins - some of which havebeen intimately involvedin the modifications discussed above - and a protein complex called the nuclear pore complex (NPC). Transport through the NPC allowsflowof proteins and RNA species to move in both directions andis mediatedbya number ofproteins.This process can be usedto selectively regulate the transport of various transcripts depending on which proteins associate with the transcript in question. This means that not all transcriptsare treatedequally by the NPC - depending on modification state and the proteins that have associated with a specific species of RNA it canbe movedeither more or less efficiently across the nuclear membrane.Sincethe rate of movement across the pore will influence the abundance of mature transcript thatis exportedinto the cytosol for translation export control is another example of a step in the process of gene regulation that canbe modulated. In addition, recent research has implicated interactions between the NPC and transcription factors inthe regulation oftranscription initiation, likely through some mechanism whereby the transcription factors tether themselves to the nuclearpores. This last exampledemonstrateshow interconnected the regulation of gene expression is across the multiple steps of this complex process.

We know many additional details of the processes described above to some level of detail, but many more questions remain to

be answered

. For the sake of Bis2a it

is sufficient

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

generally

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

in some way

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

here

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

pretty

smart about that!). Alternatively, you may raise some brand new question that no one has thought of yet.

Control of Protein Abundance

After a mRNA has been transported to the cytoplasm,it is translatedinto protein. Control of this processis largely dependenton the RNA molecule. As previously discussed, the stability of the RNA will have a large impact on its translation into a protein. As the stability changes, theamount oftime that it is available for translation also changes.

The initiation complex and translation rate

Like transcription,translation is controlled by proteinscomplexes of proteins and nucleic acids that must associate toinitiatethe process. In translation,one of the first complexes that must assembles to start the process is referredto as the initiation complex. The first protein to bind to the mRNA that helpsinitiatetranslationis calledeukaryotic initiation factor-2 (eIF-2). Activity of the eIF-2 protein is controlled by multiple factors. The first iswhether or notitis boundto a molecule of GTP. When theeIF-2is boundto GTPit is consideredto bein an active form. TheeIF-2 protein bound to GTP can bind to the small 40S ribosomal subunit. When bound, theeIF-2/40S ribosome complex, bringing with it the mRNA tobe translated, also recruits the methionine initiator tRNA associates.At this point, whenthe initiator complexis assembled, the GTP is hydrolyzed into GDP creating an"inactive form ofeIF-2 thatis released, along with the inorganic phosphate, from the complex. This step, in turn,allows the large 60S ribosomal subunit to bind and tobegin translatingthe RNA. The binding ofeIF-2 to the RNA further controlled by protein phosphorylation. WheneIF-2 is phosphorylated, it undergoes a conformational change and cannotbindto GTP thus inhibiting the initiation complex from forming - translation is therefore inhibited (see the figure below). In the dephosphorylatedstateeIF-2 can bind GTP and allow the assembly of the translation initiation complex as described above. The ability of the cell therefore to tune the assembly of the translation invitation complex via a reversible chemical modification (phosphorylation) to a regulatory protein is another example of how Nature has taken advantage of even thisseeminglysimple step to tuned gene expression.

An increase in phosphorylation levels ofeIF-2 hasbeen observedin patients with neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s. What impact do you think this might have on protein synthesis?

Chemical Modifications, Protein Activity, and Longevity

Not to

be outdone

by nucleic acids, proteins can also

be chemically modified

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

length of

time they exist in the cell. Sometimes these modifications can regulate where a protein

is found

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.

In addition to

regulating the function of the proteins themselves, if these changes occur on specific proteins they can alter epigenetic accessibility (

in the case of

histone modification), transcription (transcription factors), mRNA stability (RNA binding proteins), or translation (

eIF

-2) thus feeding back and regulating various parts of the process of gene expression.

In the case of

modification to regulatory proteins, this can be an efficient way for the cell

to rapidly change

the levels of specific proteins in response to the environment by regulating various steps

in the process

.

The addition of an ubiquitin group has another function - it marks that protein for degradation. Ubiquitin is a small molecule that acts like a flag

indicating

that the tagged proteins should

be targeted

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

be recycled

. Ubiquitination (the addition of a ubiquitin tag), therefore helps to control gene expression by altering the functional lifetime of the protein product.

Proteins with ubiquitin tags are marked for degradation within the proteasome.

In conclusion, we see that gene regulation is complex and thatit can be modulatedat each step inthe process ofexpressing a functional gene product.Moreover, theregulatory elements that happen at each step can act to influence other regulatory steps both earlier and later in the process of gene expression (i.e. the process of chemically altering a transcription factor can influence the regulation of its own transcription many steps earlier in the process). These complex sets of interactions formwhat are known asgene regulatory networks. Understanding the structure and dynamics of these networks is critical for understanding how different cells function, the basis fornumerousdiseases, developmental processes, and how cellsmake decisionsabout how to react to the many factorsthatare in constant flux both inside and outside.