Cell Biology

III. DNA and Protein Synthesis

Much of the energy harvested by a cell is used to make proteins. However, in order to understand protein synthesis, we must first describe the structure of DNA. DNA is the 'recipe' for proteins, so we need to take a little detour on our jounrey of how a cell works to describe the structure of DNA and chromosomes. DNA is the genetic material in all forms of life (eubacteria, archaea, protists, plants, fungi, and animals). Those quasi-living viruses vary in their genetic material. Some have double-stranded DNA (ds-DNA) like living systems, while others have ss-DNA, ss-RNA, and ds-RNA. RNA performs a wide array of functions in living systems. Many of these functions have only been discovered in the last few years.

A. DNA and RNA Structure

DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are nucleic acids - polymers consisting of a linear sequence of linked nucleotide monomers. We will describe the structure of the monomers first, and then describe how they are linked into linear polymers. Finally, we will describe the double-stranded structure of ds-DNA.

1. The monomers are "nucleotides"

three components:

- Pentose (5 carbon) sugar: either ribose (RNA) or deoxyribose (DNA). The carbons are numbered clockwise. The difference between the sugars is that ribose has an -OH group on the 2' carbon, whereas deoxyriboes has only 2 H groups and thus is "deoxygenated" relative to ribose. BOTH sugars have an -OH group on the 3' carbon, which will be involved in binding. The 5' carbon is a sidegroup off the ring.

- Nitrogenous Base: each nucleotide has a single nitrogenous base attached to the 1' carbon of the sugar. This nitrogenous base may be a double-ringed structure (purine) or a single ringed (pyrimidine) structure. The purines are adenine (A) and guanine (G). The pyrimidines are thymine (T), cytosine (C), and uracil (U). DNA nucleotides may carry A, G, C, or T. RNA nucleotides carry either A, G, C, or U.

- The third component of a nucleotide is a phosphate group, which is attached to the 5' carbon of the sugar. When a nucleotide is incorporated into a chain, it has a single phosphate group. However, nucleotides can occur that have two or three phosphate groups (dinucleotides and trinucleotides). ADP and ATP are important examples of these types of molecules. In fact, the precursors of incorporated nucleotides are trinucleotides. When two phosphates are cleaved, energy is released that can be used to add the remaining monophosphate nucleotide to the nucleic acid chain.

2. Polymerization is by 'dehydration synthesis'

As with all other classes of biologically important polymers, monomers are linked into polymers by dehydration synthesis. In nucleic acid formation, this involves binding the phosphate group of one nucleotide to the -OH group on the 3' carbon of the existing chain. For the purposes of seeing how this reaction works, we can envision an H+ on one of the negatively charged oxygens of the phosphate group. Then, a molceule of water can be removed from these two -OH groups, leaving an oxygen binding the sugar of one nucleotide to the phosphate of the next.

This creates a 'dinucleotide'. It has a polarity/directionality; it is different at its ends. At one end, the reactive group is the phosophate on the 5' carbon. This is called the 5' end of the chain. At the other end, the reactive group is the free -OH on the 3' carbon; this is the 3' end of the chain. So, a nucleic acid strand has a 5' - 3' polarity.

3. Most DNA exists as a 'double helix' (ds-DNA) containing two linear nucleic acid chains.

a. the nitrogenous bases on the two strands are 'complementary' to each other, and form weak hydrogen bonds between them. A always pairs with T, and C always pairs with G. As such, there is always a double-ringed purine pairing with a single-ringed pyrimidine, and the width of the double-helix is constant over its entire length.

b. the two strands (helices) are anti-parallel: they are arranged with opposite polarity. One strands points 5' - 3', while the other points 3' - 5'. The direction of the pentose sugars and the type of reactive group at the ends of the chains show this relationship.

4. RNA performs a wide variety of functions in living cells:

a. m-RNA (for "messenger") is the copy of a gene. It is the sequence of nitrogenous bases in m-RNA that is actually read by the ribosome to determine the structure of a protein.

b. r-RNA (for "ribosomal") is made the same way, as a copy of DNA. However, it is not carrying the recipe for a protein; rather, it is functional as RNA. It is placed IN the Ribosome, and it helps to ‘read’ the m-RNA.

c. t-RNA (for "transfer") is also made as a copy of DNA, but it is also functional as an RNA molecule. Its function is to bind to a specific amino acid and incorporate it into the amino acid sequence as instructed by the m-RNA and ribosome.

d. mi-RNA (micro-RNA) and si-RNA (small interfering RNA) bind to m-RNA and splice it; inhibiting the synthesis of its protein. This is a regulatory function.

e. sn-RNA (small nuclear RNA) are short sequences that process initial m-RNA products, and also regulate the production of r-RNA, maintain telomeres, and regulate the action of transcription factors. Regulatory functions.

B. Protein Synthesis

As we've already mentioned, protein synthesis is fundamental to nearly everything a cell does. Protein channels are used to transport large molecules across the membrane. Almost all chemical reactions occuring in cells are catalyzed by protenaceous enzymes, including those involved in energy harvest, DNA replication, and cell division. Proteins perform important structural functions within cells and multicellular organisms, too; such as the histone proteins in chromosomes, the proteins in ribosomes, the collagen and elastin fibers that hold skin cells together, the collagen on which calcium and phosphate is deposited in bone, the protein myofibrils of actin and myosin in muscle cells, the neurotransmitters used for cell-cell communication between neurons, and the enzymes that digest food in the stomach and intestine of animals. So, proteins are fundamental to what cells and organisms ARE, structurally, and what they DO functionally. As you know, the genetic information determines the types of proteins a cell can make. The subset of proteins a cell actually DOES make, and the timing of WHEN they are made, is determined by what genes are "on" and what genes are "off" at a given time. This regulation of gene activity is ALSO co-ordinated by proteins - called transcription factors - that bind to DNA and promote or inhibit gene activity. So, proteins also regulate protein synthesis. Hopefully you see just how important proteins are to cells and organisms. So, the process of making these proteins is important, too. It is no surprize, then, that a lot of the energy harvested by a cell is used to make proteins. The phosphate bonds in ATP are broken, and the energy that is released is used to form new bonds between amino acids. The chain of amino acids that is produced becomes a functional protein.

1. Overview

The sequence of nitrogenous bases in a region of DNA is 'read' by a complex of enzymes that build a complementary strand of RNA. This process of reading DNA and making RNA is called 'transcription'. This is a great word for the process, as the message written in the language of nucleic acids is copied in essentially the same language - the language of nucleic acids. This RNA may be a recipe for a protein (m-RNA), or it may be an RNA that will act on its own as t-RNA, mi-RNA, si-RNA, or be complexed with proteins in the ribosome (r-RNA). Obviously, in "protein synthesis", only the m-RNA is read to make a protein. However, the other molecules all play a role. The sequence of nitrogenous bases in the m-RNA is then 'read' by a ribosome, which links a specific sequence of amino acids together into a protein based on that sequence of nitrogenous bases in the m-RNA. This process is called 'translation'. This is a great choice of a word, too. Here the sequence of information written in the language of nucleic acids is rewritten in a new language (hence, translation) - the language of amino acids.

Many of the initial RNA products have specific regions (introns) cut out of their sequence before they become functional. This step is known as "RNA processing" or "RNA splicing". Introns are present in nearly all eukaryotic RNA's, and are also in the DNA genes that encode them. Up until a few years ago, the only introns in prokaryotes had been found in t-RNA molecules of archaeans. More recently, however, introns have been found in m-RNA and r-RNA molecules of a few eubacteria and a few more archaeans. So, although they are rare in prokaryotes, we will describe a generic, simplified process of protein synthesis that includes introns and RNA processing.

In addition to splicing the RNA product of transcription, the initial protein product of translation may also be spliced and modified before it becomes functional. In eukaryotes, this protein processing often occurs in the Golgi apparatus.

The description presented here is a simple model of protein synthesis. You will learn more complex aspects of this process in Genetics.

2. The Process of Protein Synthesis

1. Transcription:

a. The message is on one strand of the double helix - the sense strand: The DNA double helix is composed of two anti-parallel complementary strands of DNA. Only one strand in a coding region ("gene") is read; this strand carries a meaningful recipe that "makes sense". This is called the "sense" strand. The other strand, limited by complementarity, is not a meaningful message - it is the "non-sense" (or "anti-sense") strand. Think about it this way. Given a meaningful message of "C-A-T" (a small furry mammal), the complementary strand is limited to the meaningless sequence of "G-T-A" (????...). As the meaningful sequence gets longer, it is even LESS likely that, just by chance, the complementary strand would be meaningful, too. Again, in all eukaryotic genes and in some rare prokaryotic ones, there will be non-functional "introns" interspersed throughout the meaningful message. The meaningful parts are called "exons". The process of transcription is continuous, so introns and exons get transcribed and these regions - if present in the DNA - will also be present in the RNA product.

b. The cell 'reads' the correct strand based on the location of the promoter, the anti-parallel nature of the double helix, and the chemical limitations of the 'reading' enzyme, RNA Polymerase. RNA Polymerase binds to the DNA at a specific sequence next to the gene, called the 'promoter'. It binds in a specific way, so it is pointed towards the gene. RNA polymerase can only create a strand of RNA in the 5' to 3' direction, adding a new base to the free -OH group of the preceeding nucleotide on the chain. So, from its position at the promoter, looking down the two strands in the gene, the RNA Polymerase can only 'read' one strand - the DNA strand that is 3'-5'. It must create a strand that is anti-parallel to the DNA' template', and it can only bind nucleotides in 5'-3' direction. So, only the 3'-5' DNA strand is read in this region, and only one RNA strand, 5'-3' is made. It is important to appreciate that this relationship is 'local'. In another region of the DNA, the promoter may be on the other side of the gene, and the other strand may be read.

c. Transcription ends at a sequence called the 'terminator'.These regions have specific sequences that destabilize the attachment of the RNA Polymerase to the DNA... it detaches and transcription stops. VIDEO
So, the process of transcription can be summarized like this: RNA Polymerase binds at the promoter and reads the sense strand of DNA. The ploymerase links together RNA nucleotides 5--> 3, in a sequence complementary to the DNA sense strand. This process is continuous, so all DNA bases are 'read', including exon and intron sequnces. This process continues until a terminator region is reached. Reading this region destabilizes the RNA polymerase. It detaches from the DNA, and transcription stops. All types of RNA (m-RNA, r-RNA, t-RNA) are made through this process.

2. Transcript Processing:

At this point in the process, the cell has read the gene and synthesized a complementary copy of strand of RNA. In all eukaryote sequences and many prokaryotic ones, this RNA molecule will have non-functional introns that need to be 'cut-out'. Enzymes cut the introns out and splice the ends together. In some cases, the introns catalyze their own excision - they are RNA molecules with enzymatic activity. These are one class of "ribozymes" - a very interesting class of molecules. There are other ribozymes that cleave other RNA molecules (not themselves) and others that catalyze other chemical reactions unrelated to RNA splicing.

In eukaryotes, the m-RNA, t-RNA and r-RNA is shunted through the nuclear membrane to the cytoplasm. In prokaryotes, there is no nucleus so the RNA is already in the cytoplasm. In all organisms, the r-RNA is complexed with proteins to form functional ribosomes. The t-RNA's bind specific amino acids.

VIDEO

3. Translation:

In this process, amino acids are linked together into a protein. The particular sequence of amino acids that are linked together is determined by the sequence of nitrogenous bases in m-RNA. This process occurs at the ribosome.

a. m-RNA attaches to the ribosome at the 5' end. The ribosome has two reactive sites. The RNA moves through the ribosome until a specific sequence of nucleotides, AUG, is positioned in the first site. Three base sequence in the m-RNA are called 'codons'. This specific codon AUG, which starts the process of translation, is called the 'start codon'. All proteins made by all life forms initially begin with methionine, and use the codon AUG..

b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds to the m-RNA/ribosome complex. This t-RNA always carries the amino acid methionine. The genetic code describes the relationship between 3-base codons in m-RNA and the amino acids they code for.

c. Binding of the t-RNA to the first site opens a second site that reads the second 3-base codon (GCC in picture at right). Another t-RNA binds here - one with the specific anti-codon sequence (CGG). This t-RNA, with this anti-codon, always binds with the amino acid alanine.

d. Now a complex series of reactions occurs. Methionine is cleaved from its t-RNA and bound to alanine (this peptide bond between amino acids forms via dehydration synthesis). The t-RNA in position 1 vacates the site, and the t-RNA in site 2 moves to site 1. This is called a 'translocation reaction'. The next 3-base codon is positioned in the second site - ready to accept the next t-RNA/ammino acid complex (for tryptophan in the picture to the right).

e. Polymerization proceeds. This process continues down the m-RNA strand, reading the message one codon (3-base "word") at a time. For each codon, a specific amino acid is added to the chain. Thus, the nucleotide sequence in the m-RNA - copied from the nucleotide sequence in the DNA gene - determines the sequence of amino acids in the protein.

f. Termination. There are some codons that have no corresponding t-RNA molecule. When these codons enter the second site, no t-RNA/amino acid is added. When the ribosome translocates, no new amino acid is added and the chain is terminated. These particular codons that stop translation are called "stop codons".

VIDEO

4. Protein Processing:

The initial protein product usually needs to be modified to become functional. These modifications are termed "post-translational modifications". First of all, the methionine is usually cut off - this relieves an important constraint on the structure of functional proteins... functional proteins DON'T all start with methionine! Then, the protein may be spliced, or it may be bound with a sugar group (glycoprotein), lipid (lipoprotein), nucleic acid (nucleoprotein), or another protein (quaternary protein). In eukaryotes, much of this processing occurs in the Golgi apparatus.

3. Regulation of Protein Synthesis

Aside from somatic mutations, all the cells in a multicellular organism are genetically identical. So, the cells in your retina, bone, muscle, and stomach lining all contain the same genes. These cells perform different functions because they are reading different genes and making different proteins. Your muscle has the gene for rhodopsin (a photoreceptive pigment produced in the retina), but that gene is not transcribed in muscle cells. In contrast, retinal cells have the genes for the muscle proteins actin and myosin, but these genes are not transcribed. So, cell specialization and the developmental process by which cells specialize from the fertilized egg occurs by regulating this process of protein synthesis. Regulation can occur at each of the steps described above.

1. Regulation of Transcription:

The process of transcription is regulated in several ways. First, the RNA polymerase can be blocked from the promoter. This can happen because the gene is bound to histones in a nucleosome, or is in a region of condensed 'heterochromatin', or because other proteins called 'transcription factors' have bound to the DNA - either at the promoter or between it and the gene, blocking the polymerase's route. However, the binding of other transcription factors can increase the affinity of the RNA polymerase for the promoter - increasing the probability of transcription. Again, these transcription factors are proteins encoded by other genes, and affected by other cellular processes. In this way, the action of a gene can be co-ordinated with the activity of other genes in a complex and interdependent manner. In addition, environmental cues from outside the cell can, through signal transduction, affect the activity of transcription factors and turn genes on or off. So, an organism can respond genetically to environmental cues.

2. Regulation of Transcript Processing:

The production of a protein can be affected at the processing stage. mi-RNA's and si-RNA's are small RNA molecules encoded by their own genes. These molecules can bind to m-RNA and effectively block correct splicing. This turns off production of the correct protein. In some cases, an initial m-RNA can be spliced two ways, creating two different functional products (and eventually two different proteins) depending on the pattern of cleavage. So, one gne may code for different proteins in different cells or tissue types.

3. Regulation of Translation:

One way that differential splicing can affect protein production is by changing the location of stop codons. For example, suppose a stop codon occurs at the beginning of an intron. Then, suppose that the intron is spliced incorrectly, after the location of this stop codon. Now, the resulting functional m-RNA has a stop codon where it didn't before; and translation will be terminated prematurely and no functional protein will be produced.

4. Regulation of Post-Translational Modification:

Initial protein products can be cleaved in different ways to produce different proteins, too.

So, through all of these mechanisms, protein synthesis can be stopped or stimulated, and the product can be modified. Again, all of these regulatory pathways can be affected by environmental factors or the proteins or mi/si-RNA's produced by other genes. So, gene activity is affected by other things happening in the cell (turning other genes on and off) , in other cells of the organism (through the production of hormones that act as signal transducers), or environmental factors outside of the organism acting directly on this gene, on other genes in this cell, or on other cells..

Cell division is the process of producing two functional 'daughter' cells from one ancestral 'parental' cell. In order for both of the daughter cells to have the full functional repertoire of the original parental cell, they must be able to make the full complement of proteins that the parent cell makes. In order for this to happen, they must both receive the full complement of genetic information (DNA) in the parental cell. Hmmm.... how can they BOTH get the FULL COMPLEMENT of genetic information in the parental cell? Well, in order for this to happen, the parental cell must duplicate its DNA prior to cell division. This process of DNA replication produces two full complements of genetic information. Then, this genetic information must be divided evenly, in an organized manner, to insure that both daughter cells get the complete complement of information (and not a duplication of some information or an omission of other information). Cells that receive an incomplete complement of genetic information will not be able to make all the proteins the parental cell made, and may not be able to survive. So, again, DNA replication and the process of mitosis are of great selective, adaptive value. Only cells that replicate and divide their genetic information evenly, with only minor errors or inconsistencies, will be likely to survive. These survivors will pass on the tendancy to replicate and divide their genetic information evenly, as well. So, there is very strong selection ( a very large selective advantage) for correct DNA replication and equal chromosomal allocation during mitosis.

These processes of DNA replication and mitosis are only two stages in the life of a cell. To place them in context, it's useful to consider the full life of a cell, from it's production by the division of its parental cell through to its own division.

D. Mitosis and the Cell Cycle

1. Interphase - the 'interval' between divisions

a. G1

Our cell's life begins. That's sort of a funny way to put it, because it seems to suggest that it is something new; yet all of its constituents were part of the original parental cell. It is more truly "1/2 an old cell with a full complement of DNA". Nevertheless, it is an independent entity. In most protists, binary fission of the mitochondria and chloroplasts occurs concurrently with the division of the nucleus during mitosis, so the daughter cells have 'new' organelles, too. But in most multicellular organisms, the allocation of organelles is largely a random process based on how they are distributed in the cytoplasm during division. Then, the organelles divide and 'repopulate' each daughter cell in G1.

The cell is roughly 1/2 the size of the original parental cell. To grow to its appropriate size, it must synthesize new biological molecules - and that means making the enzymes that will catalyze those reactions. So, the DNA unwinds to the 'beads on a string' level, and the genes between histones are available for transcription. When the DNA is unwound ('diffuse'), separate chromosomes cannot be seen with a light microscope. Rather, the nucleus stains a uniform color except for one or several dark regions called 'nucleoli' (singular = nucleolus). These are areas were large amounts of r-RNA are being synthesized and complexed with ribosomal proteins into functional ribosomes. The ribosomes are exported from the nucleus to the cytoplasm, where they will anchor to endoplasmic reticulum or the cytoskeleton.

Indeed, the G1 phase of a cell's life is the most metabolically active period of it's life. It is growing in size, and producing the proteins appropriate for its tissue type. Most cells in multicellular organisms specialize during this period. Cells with very specific structural adaptations to their specialized tissue type - like neurons with long axons and muscle cells crammed with linear microfilaments - often remain stalled in this stage after they become specialized; they do not divide again. In this case, this stalled 'permanent' G1 phase is referred to a G0 ("G-nought').

b. S

The S phase of the cell cycle is when DNA replication occurs. The chromosomes are diffuse during this stage, as well, so the enzymes (DNA polymerases) that replicate the DNA can access the helices. Each double helix is separated, and the single strands are used as templates for the formation of new helices on each template - changing one double helix into two. Terminology becomes a bit ambiguous here. A DNA double helix is equivalent to a "chromatid". A chromosome may have one chromatid (in its unreplicated form) or two chromatids (in its replicated form). DNA replication is a rather complicated process described in more detail below. The transition from the G1 to the S phase is a very critical stage in a cell's life cycle, signalling the cell's progression towards division. In eukaryotes it is called a 'restriction point'. Once the S phase begins, the cell will proceed through to mitosis. This transition is orchestrated by a complex interplay of transcription factors that regulate the activity of "cell division cycle genes". These genes produce cyclin proteins that vary in concentration through the cell cycle. They bind with 'cyclin-dependent kinases' and these cdk-cyclin complexes activate transcription factors that initiate the next phase of the cell cycle.

c. G2

After DNA replication is complete the cell goes through another rapid period of growth in preparation for mitosis. The DNA is checked again for damage caused and errors made during DNA replication. Once again, p53 inhibits the transition to the mitotic phase, providing time for this repair to take place. In cancer cells with mutations in p53, the G2 phase may be nearly eliminated, with the cell proceeding directly from DNA replication to mitosis. CDK's bind to new cyclins, and these complexes active a different set of proteins that initiate mitosis.

2. Mitosis

The process of mitosis can be summarized as follows: the chromosomes condense, making it easier to divy them up evenly. The replicated chromsomes are aligned in the middle of the cell by cytoskeletal fibers. Each chromosome consistes of two identical double helices, called chromatids. During the process of mitosis, these chromatids separate from each other, and one double-helix from each chromosome is pulled to each end of the cell. The membrane and cytoplasm are divided and the nuclear membrane reforms around the chromosomes in each daughter cell. We will look at this process in more detail, below.

Mitosis is a continuous process of chromosome condensation, chromatid separation, and cytoplasmic division. This process is punctuated by particular events that are used to demarcate specific stages. This process was first described by Walther Flemming in 1878, we he developed new dyes and saw 'colored bodies' (chromo-somes) condensing and changing position in dividing cells. He also coined the term 'mitosis' - the greek word for thread - in honor of these thread-like structures.

1. Prophase: The transition from G2 to Prophase of Mitosis is marked by the condensation of chromosomes.

2. Prometaphase: The chromosomes continue to condense, and the nuclear membrane disassembles. The microfibers of the spindle apparatus attach to the kinetochores on the replicated chromsomes.

3. Metaphase: The spindle aranges the chromosomes in the middle of the cell.

4. Anaphase: The proteins gluing sister chromatids together are metabolized, and the sister chromatids are pulled by their spindle fibers to opposite poles of the cell. It is important to appreciate that these separated chromatids (now individual, unreplicated chromosomes) are idnetical to one another and identical to the orignial parental chromosome (aside from unrepaired mutations).

5. Telophase: The cell continues to elongate, with a concentrated set of chromosomes at each end. Nuclear membranes reform around each set of chromosomes, and the chromosomes begin to decondense.

6. Cytokinesis: Cytokinesis is sometimes considered a part of telophase. In this stage, the cytoplasm divides. In animal cells, the membrane constricts along the cell's equator, causing a depression or cleavage around the mid-line of the cell. This cleavage deepens until the cells are pinched apart. In plants, vesicles from the golgi coalesce in the middle of the cell, expanding to form a partition that divides the cell and acts as a template for the deposition of lignin and cellulose that will form the new cell wall between the cells.

As a consequence of this process, two cells are produced from one parental cell, each having a complete complement of genetic information - a copy of each original chromosome that was present in the parental cell. Each of these cells now begins the G1 phase of interphase.

STUDY QUESTIONS:

1) Diagram the parts of an RNA nucleotide.

2)  Show how two nucleotides are linked together by dehydration synthesis reactions.

3) Why does the purine - pyrimidine structure relate to the complementary nature of double-stranded DNA?

4)  Draw a DNA double helix, showing three base pairs and the antiparallel nature of the helices.

5) Describe the higher levels of eukaryotic chromosome structure, including the terms nucleosome and solenoid.

6) What 'cues' determine where transcription will start and stop?

7) What are introns and exons, and how are they processed?

8) Describe translation: what is read, what is produced, and how is the 'genetic code' involved?.

9) How is a polypeptide modified after translation to become a functional protein?

10) The process of protein synthesis and the universal genetic code provides one of the dramatic pieces of evidence of a common ancestry to all life. Explain why.

11) Draw the cell cycle, labeling each stage and highlighting the main event in each stage.

12) Draw a chromsome before and after replication; use the terms chromosome and chromatid.

13) Draw a cell, 2n = 6, and show each of the stages of mitosis. Write a brief description of the events of each stage.