DNA expression and regulation– protein synthesis

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DNA, RNA and ribosomes, in that order, are essential components in the synthesis of proteins. DNA contains the information necessary not only for reproduction, but also for daily cell growth and maintenance. Messenger RNA carries the information to the ribosomes, themselves containing RNA. With the help of yet another kind of RNA, the ribosomes assemble the proteins. All this depends on gene regulation.

The use of DNA to initiate protein synthesis is called DNA expression.

This sequence of events is summed up in the so-called central dogma of molecular biology, often paraphrased as “DNA makes RNA and RNA makes protein.” More precisely, DNA is transcribed inside the nucleus to make mRNA, which is expelled from the nucleus to the cytoplasm, where it is translated to protein by ribosomes.

DNA –> transcription (nucleus)→ mRNA→ translation (ribosome)→ protein.

The recipe is expressed in “bytes” of three nucleobases; one three-base byte is referred to as a code-word. When transcribed to its complementary form in mRNA, it is called a codon. Since each base can have one of four values (C, G, A or T, in DNA), the codon can take on 64 values.

RNA transcription

The enzyme which does the work of “reading” a gene on the DNA and building a corresponding gene of RNA is called RNA polymerase1)In fact, there are several forms of RNA polymerase, but that complexity is well beyond the scope of this document.. There are at least four types of RNA and transcription makes them all. For protein synthesis, the RNA constructed is called mRNA, or messenger RNA. The DNA recipe begins with a sequence called the promoter. RNA polymerase contains a complementary sequence which binds to the promoter and launches transcription. As will soon be seen, transcription is started only if it is allowed by gene regulation. RNA polymerase unwinds a part of the DNA chain and reads code-words, starting with the promoter. As it reads the DNA, it constructs a complementary chain, called pre-mRNA, from nucleotides. It is complementary in the sense that if the DNA contains a C (or A or G or T) then the pre-mRNA contains a G (or U or C or A – remembering that RNA replaces T by U).

The raw materials RNA polymerase uses to construct RNA are nucleoside triphosphates (NTPs). Two of its phosphate groups contain a significant amount of energy from ATP. This energy is used to bond the nucleotides together to form RNA.

The RNA polymerase moves along the DNA, unwinding sections as it goes, reads the code words and assembles the appropriate pre-mRNA codons from NTPs. The separated DNA strands recombine in its wake. Eventually, it reaches a transcription-terminator sequence in the DNA and ends transcription. It now has gone through three steps, known as initiation, elongation (of the produced pre-mRNA) and termination. The pre-mRNA then is released into the nucleoplasm.

Splicing mRNA, from Openstax College

Splicing mRNA, from Openstax College

Before leaving the nucleus, the pre-mRNA must be cleaned up. This is needed because DNA contains non-coding, or junk, sequences. The codons which should be kept are called exons (like “expressed”) and those which should be deleted are called introns (like “interrupted”).2)I would have preferred for exon to mean “exit”, to be got rid of, and intron to mean “in”, as kept in, but some contrary biologist decided otherwise. He could at least have taken a vote! Small particles called “snurps” (for snRNPs, or small nuclear ribonucleoproteins), made up of RNA and proteins, bind together to form spliceosomes, which remove introns and splice the exons back together again, resulting in a cleaned-up form of mRNA.3)Are you wondering how the snurps can recognize the introns an exons? So am I. All I can say is that it is quite complicated and has something to do with methylation of the DNA strands. It is currently not completely understood why there are introns at all, but there are indications that they may be of importance.

The mRNA is then moved out of the nucleus for the next step.

Protein synthesis – translation

After the mRNA leaves the nucleus, it is used to provide the input data for the synthesis of proteins. This takes place on ribosomes.

There are two sorts of ribosomes in eukaryotic cells, depending on their location.

  • Free ribosomes float in the cytoplasm and make proteins which will function there.
  • Membrane-bound ribosomes are attached to the rough endoplasmic, reticulum, which are what makes it look “rough”. Proteins produced there will either form parts of membranes or be released from the cell.

In most cells, most proteins are released into the cytoplasm.

Ribosomes are made of ribosomal RNA, or rRNA (one more kind of RNA), and proteins. They are constructed within the nucleolus as two subunits, which are released through the nuclear pores into the cytoplasm.

In addition to the mRNA and the ribosome subunits, a method Is needed for supplying the appropriate amino acids to be linked by peptide bonds to make up the protein or enzyme being constructed. Enter still one more kind of RNA, transfer RNA, or tRNA.

A molecule of tRNA is a molecule of RNA folded into a double strand with loops which give it a precise 3-dimensional shape. It has on one end a binding site (adenylic acid) for an amino acid and, on the other, an anticodon, a site to match the complement of the codon on mRNA. A tRNA molecule is “charged” with an amino acid molecule by one of 20 types of tRNA-activating enzyme, or aminoacyl-tRNA synthetase, each one specific to a particular amino acid. Each type of aminoacyl-tRNA synthetase has a specific shape, which means it can only join with and service the corresponding tRNA molecule. It uses energy from ATP to covalently bond the tRNA with the appropriate amino acid from molecules in the cytoplasm. Such tRNA molecules, carrying an amino acid, are called aminoacyl tRNA.

The ribosome itself contains three spaces. It reads in the mRNA strand, like computers of my youth read in paper tape, so that it crosses the spaces as follows:

  • the mRNA enters at the A-site;
  • the current peptide element Is added to the growing chain at the P-site and the peptide chain exits from here;
  • the mRNA exits from the E-site.

Initially, the ribosome subunits are floating independently in the cytoplasm or attached to the RER.

The initiation of translation begins when the small ribosome subunit binds to the first ribosomal binding site on an mRNA strand. Initially, the tRNA carrying methionine, the amino acid indicated by the mRNA START codon, binds its anticodon to the mRNA binding site. Then the large ribosome subunit binds to the small, so the ribosome is now complete with the first tRNA in the P-site and the second codon of the mRNA in the A-site.

Gene translation in the ribosome, from Openstax College

Gene translation in the ribosome, from Openstax College

The process then pursues the elongation stage of translation. The aminoacyl tRNA for the codon in the A-site is carried in, so the first two amino acids are now in the P and A sites. The ribosome then catalyzes the formation of a peptide bond between these two amino acids. The ribosome then moves the mRNA so the first amino acid enters the E-site, the second enters the P-site, and a new one, the third, enters the A-site. It continues like that until a STOP codon enters the A-site and brings about termination of translation and release of the completed peptide chain.

All these steps of transcription and translation require energy, so protein synthesis is one of the most energetically costly of cell processes. Much of this energy is used to make enzymes essential to the functioning of the cell.

Once part of a strand of mRNA has left one ribosome, it can enter another. One strand may be in 3 to 10 ribosomes at once, in a different step of translation in each one. Such clusters of ribosomes translating the same mRNA strand are called polyribosomes.

Regulation of gene expression

Every cell in an organism has the same complete genome in its nucleus and so has access to all the same protein “recipes”. But heart cells should not produce proteins used only by the liver and no cell should produce proteins in quantities beyond what it can use. Controlling which proteins to express and when is called regulation. Note that this is one more instance of communication in the body, telling genetic machinery when and what to express.

Regulation of prokaryotic cells

Regulation in prokaryotic cells is relatively simple, as there is no nucleus. so transcription and translation take place almost in the same place and at the same time. Regulation in prokaryotic cells, though, almost always concerns transcription.

An example from a prokaryotic cell will show how this works – and introduce some new terminology.

The bacterium E. Coli normally uses glucose for energy. But if glucose is absent and lactose is present, it can use the latter sugar. The proteins necessary for the use of lactose are controlled by a sequence of genes called the lac operon. The operon includes not only the necessary genes, but, at the beginning, a promoter which indicates the beginning of the operon and is the site where RNA polymerase binds to begin the transcription. In between the promoter and the set of genes, of which there may be any number, is a sequence called the operator, which is where DNA-binding genes bind to regulate transcription.4)Look out for the terminology: Sean B. Carroll refers to the operator as a genetic switch, a term we will meet with in the disussion of regulation in eukaryotes.

When no lactose is present, a protein called the lac repressor is bound to the operator and the state of the lac operon is “off”. (Figure.) This is because the repressor blocks access to the rest of the operon.

The gene for the lac repressor is a constitutive gene: It is always expressed because it is the recipe for an essential protein. On the other hand, a regulated gene, is expressed selectively.

Regulation of the lac operon, from Openstax College

Regulation of the lac operon, from Openstax College

The lac repressor has a second, allosteric, binding site. When lactose is present, an isomer of lactose binds to the allosteric site of the repressor, which causes it to change its form and unbind from the operator. The lac operon is now in the “on” state. This form of regulation is called induction: Lactose is said to be the inducer of the lac operon and acts through the allosteric site of the lac repressor. Transcription now occurs, but slowly, because some glucose still may be present. But are the proteins mapped by the lac-digesting genes needed? In other words, is glucose very lacking?

The answer to that question and the second part of this process depends on the presence of glucose and is regulated by a second DNA-binding protein, CAP (catabolite activator protein). CAP is also an allosteric protein with one DNA-binding site and one allosteric site which binds to cyclic AMP (cAMP). CAP is only active when it is bound to cAMP. You guessed it, cAMP levels are high when glucose levels are low.5)If we go one step back, we see that glucose binds to an allosteric site on the enzyme adenylate cyclase, which makes cAMP from ATP, and disables it. So lack of glucose stimulates production of cAMP, which binds to CAP, which binds to the promoter to enhance synthesis. In that case, cAMP-CAP binds to the promoter and enhances transcription of the genes. So lactose can be considered the “on-off” switch for transcription of genes for lactose-digestion and cAMP-CAP, the “volume control”.

Regulation of eukaryotic cells

In eukaryotic cells, transcription occurs inside the nucleus and translation outside, so mRNA is shuttled across the nuclear membrane in between the two processes. Regulation in eukaryotic cells therefore may take place inside or outside the nucleus or at any step in the expression pathway, including control of access to the gene in the DNA, control of transcription, pre-mRNA processing, mRNA lifetime and translation, and modification of the final proteins. Even the activity levels of enzymes which facilitate expression can be controlled.

Pre-transcription regulation

Inside the nucleus, histones, around which chromatin is wound to make nucleosomes, can wind or unwind to change spacing of the nucleosomes and thereby allow or deny access to genes. This process is a form of epigenetic regulation.6)Look out, epigenetic regulation is used for somewhat different notions, too, and they are not all necessarily so. Since histones are positively charged and DNA, negatively, modifying the charge by adding chemical “tags” to either modifies the configuration of the DNA.

Transcription regulation

The most frequent regulation of expression in eukaryotes is during transcription. Control of transcription by the prokaryotic lac operon is a relatively simple process: In order to begin transcription of a gene, RNA polymerase must bind with the gene’s promoter but cannot do so if a lac repressor is bound to the operator region which follows the promoter on the gene.

Eukaryotic gene expression is regulated similarly, but with far more of everything. The single operator is replaced by a number of regulatory sequences, sometimes referred to as switches, which may be almost anywhere on the DNA strand, even far from the gene. The repressor is replaced by a slew of regulatory proteins called transcription factors. Gene expression is controlled when transcription factors bind to regulatory sequences. The existence of multiple switches for each gene allows the gene to be more than once and in different places; each place may activate a different switch.

There are two types of transcription factors.

  • general transcription factors affect any gene in all cells and are part of the transcription-factor complex;
  • regulatory transcription factors affect genes specific to the type of cell.

The two types of transcription factors work together with three types of regulatory sequences.

  • promoter proximal elements are, of course, near the promoter and turn transcription on;
  • enhancers are far from the regulated genes or in more than one place and also turn the transcription on;
  • silencers are also far away from the regulated genes but turn transcription off.

Activator transcription factors bind to enhancers to promote expression, repressor transcription factors bind to silencers to decrease expression.

The promoter in eukaryotic cells is more complex too, The basal promoter begins with the TATA box, recognized by its beginning which contains the seven-nucleotide sequence TATAAAA, followed by a set of transcription factors brought together by coactivator proteins and called the transcription factor complex. RNA polymerase only binds to the transcription-factor complex.

The whole set of transcription factors is summed combinatorially to determine whether or how much the gene will be expressed. Selective promotion or inhibition at combinations of these sites can therefore bring about tissue-specific gene expression. Each tissue type may have its own specific enhancer or silencer sequence for the same gene. For instance, the neuron-restrictive silencing element (NRSE) is a repressor which prevents genes from being expressed in any cells which are not neurons. In addition, environmental changes may bring about different gene expression according to current, perhaps temporary needs.

Transcription factors in eukaryotic cells

Transcription factors in eukaryotic cells7)Author’s own work

The above figure shows the case of an enhancer bound by activator transcription factors.8)The bobby-pin curl is an idealization; DNA shapes are far more complex than that. The enhancer, on the left, originally is quite far from the promoter, until DNA bending causes it to change its shape, allowing the enhancer to come in contact with the promoter and the rest of the transcription-initiation complex.

Since transcription factors are proteins, they also are coded by genes and these genes are regulated in turn by other transcription factors.9)Yes, i’s transcription factors all the way down, back to that original, unique zygote. But it depends on the environment too. Much research is being done on this subject. Transcription factors and signaling elements coded by some of these genes make up the genetic toolkit, as we will see in a moment.

Splicing regulation

In between transcription and translation, proteins may interfere with spliceosomes to modify splicing of pre-mRNA. Different intron selections can allow different mRNAs to be produced from the same pre-mRNA, a phenomenon known as alternative splicing.

Pre-translation and translation regulation

Yet another type of RNA, very short-stranded microRNA or miRNA, can bind with complementary mRNA before it is translated and promote repression of its translation. miRNA associates with RISC (RNA-induced silencing complex) to degrade mRNA.

Other proteins, RNA-binding proteins (RBPs), can bind with the 5′ cap or the 3′ tail of the mRNA and either increase or decrease its stability.

Phosphorylation or attachment of other chemicals to the mRNA protein initiator complex also inhibit translation.

Similar bindings may take place on the protein after translation and modify its stability, lifetime or function. RNA does not hang around forever, but eventually is degraded and is no longer functional. So controlling its lifetime is another way of regulating its activity.

The development genetic toolkit – what evo devo tells us

Development, especially embyronic development, is the means by which a genotype, a set of genes, becomes a phenotype, a particular living organism. Although mutation works on genes, natural selection works on phenotypes. So development and evolution work hand in gene, so to speak, and the branch of biology which studies them together is called “evo-devo“.

The major discovery of evo-devo is the collection of genes known as homeoboxes, the best known of which is made up of Hox genes. Many animals have a disposition of body parts along an axis, such as the antennae, wings and legs along the body axis of a fruit fly, or the existence or not of ribs along the vertebrae of a vertebrate animal. It turns out that the choice of body part at each segment along the axis is governed by a single gene. These “master” genes control the developmental differentiation of, for instance, a fruit fly’s serially homologous body parts10)The front legs of a cat and our arms are considered homologous body parts. Structures along a body axis, similar but different, are called serially homologous with respect to each other.; in simpler terms, its body pattern.

Of the approximately 1000 or so base pairs constituting each of these genes, a subset of 180 pairs, coding for 60 amino acids, is very similar to such a subset in other “master” genes; otherwise, the genes are different. The 180 base pairs are called the homeobox; the proteins they express, the homeodomain.

Homeobox genes code for transcription factors which control gene expression during development. Since the proteins discussed so far change the cells they modulate into something else, they are called homeotic11)Homeosis the transformation of one organ into  another. transcription factors and their genes are homeotic genes. The protein domain they express is therefore a homeotic domain. The genes of this particular family are called Hox genes. Other homeobox families also exist, as we will see a few in a moment. Hox genes are just one family of them.12)I can not find a precise statement of how many.

Homeotic genes occur in clusters, with the genes of a cluster in the same order as that of the body segments they control. Since the genes are ordered in the clusters, it is possible to change the gene at the antenna position on a fruit fly to a leg gene and a leg develops at this position on the fly.

Hox genes specify the development of human arms and legs as well as of fruit-fly legs and antennas. Hox genes in vertebrates are responsible for the identity of vertebrae, which ones make ribs or fuse to form the sacrum. Modifying them in mouse genes can cause ribs to form where they should not or not form where they should. Hox genes are sufficiently similar that introduction of mouse Hox genes into a fly can cause the growth of the indicated organ — in fly format. They also control the very different serial structure of snakes.

It is remarkable that quite similar homeodomains have been found in almost all animals. Such conservation of homeobox genes across species shows that embryonic development of most animals, fungi and plants is controlled by approximately the same genes. They must have been around since animals diverged from each other over 500 Mya. The original Hox gene was duplicated and then each copy took on slightly different functions. Subsequent duplications and modifications have led to the diversity of animals today. Comparison of the genes can contribute to building at least a partial tree of life.

The set of different families of homeobox genes conserved across species are considered to be members of what biologists call the genetic toolkit. The toolkit is common to almost all animals, with only little variation from one to another. It contains genes not only for transcription factors, but also for various molecules which are signaling elements. They play important roles in embryonic development, or embryogenesis.

In other animals also, the genes exist in clusters, with the gene order in the cluster corresponding to that of the organism’s parts. Different Hox genes, being similar but slightly different, bind to different regulatory sequences on DNA and therefore regulate different genes. One homeobox protein may regulate many genes and a number of homeobox proteins may work together to refine selection. Because of this possibility of multiple binding, a small change in activation of toolkit genes can bring about a large change in the phenotype. So the genetic toolkit may explain development more simply than if all genes had to be specific to each different part, location and development time of an organism.

Toolkit genes themselves have multiple switches. Switches are the means by which a relatively limited set of toolkit genes may be used differently in different regions, or even different animals, or at different times in embryonic development – which furnishes material for evolution.

A specific bodily environment (liver, heart, blood, …) contains some set of organic molecules specific to that environment. These molecules or a sub-set thereof will serve as transcription factors to activate a particular sub-set of the toolkit. In other words, the environment chooses which tools to use.13)I see this nicely in an analogy with a computing program. A program to construct an organism will contain a higher-level library of homeobox routines for making, say, eyes or legs. Another library will contain the specific routines for that organism. A mouse organism will pass control at a specific place to a Pax-6 gene which will pass control to the appropriate eye routine. The proteins expressed by toolkit genes will activate or suppress expression of body-part proteins at that place and time.

Environmental molecules ==> toolkit proteins ==> body parts

Each arrow indicates that the object to the left switches on expression of the object to the right.

Some terminology helps to understand the evo-devo literature.

  • Transcription factors are proteins and so are not on the DNA string, therefore not on the same molecule as the DNA which is regulated. They therefore are called trans-acting regulatory elements (TRE).14)In Latin, “cis” means “this side of” and “trans” means “the other side of”. Think of cis-Alpine (this side of the Alps) and trans-Alpine.
  • Switches are on the same string of DNA as the regulated gene and are called cis-acting regulatory elements (CRE).

So one can say that TREs bind to CREs to regulate gene expression.

Let’s resume:

  • A homeobox is a sequence of genes about 180 base-pairs long, corresponding to about 60 amino acids, which encode a protein domain which consists of transcription factors for genes. Homeoboxes are found, for instance, in bacteria, fruit flies, mice, frogs, cows and humans. There are different kinds, or families, of homeoboxes.
  • A homeodomain is a protein domain corresponding to a family of homeobox genes.15)A domain is a conserved part of a protein sequence which can exist independently of the rest of the protein chain.  Homeodomains can be seen as the building blocks of development and evolution.
  • The set of homeoboxes comprises the genetic toolkit.
  • Hox genes are a subgroup of homeobox genes. They occur in similar forms within genes for morphogenesis in animals, fungi and plants. The objects transcribed are regulated by a common homeobox, but correspond in their nature to the specific species.16)Some authors use the term homeobox only for Hox genes.

The following table lists just a few of the homeobox families and the organism components they regulate.

Protein name Penotype regulated
Hox body regions (e.g., head, thorax or abdomen)
Pax-6 eyes
Distal-less (Dll) limbs
Sonic organogenesis (tissue patterns)
Ulrabithorax (Ubx)  represses insect wing formation

In all animals, there exist similar gene sequences corresponding to protein domains which are transcription factors for that animal’s version of some phenotype. A Pax-6 gene from a mouse makes an eye form in a fruit fly – a fruit-fly eye, not a mouse eye.

The existence of different levels of transcription factors also explains how a small genetic change (in a transcription factor) can bring about a relatively important change in the phenotype of the organism.

Cell differentiation

Stem cells are those which may split and form any kind of cell.17)Usually to make an identical stem cell and another cell, which may or may not be a stem cell. But once a specific type of cell is made, it can only do certain things. This is because it no longer has access to the entire recipe book (genome), but only those recipes which it needs. The cell is then said to be differentiated and the process for making it is differential gene expression. Through gene regulation, they only have access to the genes they need to fulfill their particular function. Such regulation or differentiation depends on the cell’s environment.  We have seen an example where the presence of lactose induces the expression of the lac operon.

Gene regulation can fill a book. And cell differentiation can fill another.

Continue with cell division and the cell cycle.

 

 

 

 

 

Notes   [ + ]

1. In fact, there are several forms of RNA polymerase, but that complexity is well beyond the scope of this document.
2. I would have preferred for exon to mean “exit”, to be got rid of, and intron to mean “in”, as kept in, but some contrary biologist decided otherwise. He could at least have taken a vote!
3. Are you wondering how the snurps can recognize the introns an exons? So am I. All I can say is that it is quite complicated and has something to do with methylation of the DNA strands. It is currently not completely understood why there are introns at all, but there are indications that they may be of importance.
4. Look out for the terminology: Sean B. Carroll refers to the operator as a genetic switch, a term we will meet with in the disussion of regulation in eukaryotes.
5. If we go one step back, we see that glucose binds to an allosteric site on the enzyme adenylate cyclase, which makes cAMP from ATP, and disables it. So lack of glucose stimulates production of cAMP, which binds to CAP, which binds to the promoter to enhance synthesis.
6. Look out, epigenetic regulation is used for somewhat different notions, too, and they are not all necessarily so.
7. Author’s own work
8. The bobby-pin curl is an idealization; DNA shapes are far more complex than that.
9. Yes, i’s transcription factors all the way down, back to that original, unique zygote. But it depends on the environment too. Much research is being done on this subject.
10. The front legs of a cat and our arms are considered homologous body parts. Structures along a body axis, similar but different, are called serially homologous with respect to each other.
11. Homeosis the transformation of one organ into  another.
12. I can not find a precise statement of how many.
13. I see this nicely in an analogy with a computing program. A program to construct an organism will contain a higher-level library of homeobox routines for making, say, eyes or legs. Another library will contain the specific routines for that organism. A mouse organism will pass control at a specific place to a Pax-6 gene which will pass control to the appropriate eye routine.
14. In Latin, “cis” means “this side of” and “trans” means “the other side of”. Think of cis-Alpine (this side of the Alps) and trans-Alpine.
15. A domain is a conserved part of a protein sequence which can exist independently of the rest of the protein chain. 
16. Some authors use the term homeobox only for Hox genes.
17. Usually to make an identical stem cell and another cell, which may or may not be a stem cell.