CHAPTER 5
MACHINERY
Building Smart Parts
WHEN WE HUMANS BUILD A RADIO OR A CAR OR A COMPUTER, WE ASSEMBLE INANIMATE parts using the know-how we’ve accumulated over several hundred years.When our cells build us, they use information accumulated over four billion years — and they build know-how right into the parts. The parts are “smart.” Instructions in DNA are translated into many thou- sands of ingenious devices, proteins, that do their tasks with astonishing fidelity, precision, and cooperation. Everything we do — think, laugh and cry, run and dance, conceive and give birth to children — emerges from the coordinated activities of a lively, intercommunicating society of protein molecules.
We call these proteins “machinery” because they do work that is accomplished by a simple movement. By making a subtle shift in its internal structure a protein can change its shape reversibly. If you watched one doing this all day, you’d likely be unimpressed by its I.Q. — for each protein knows only a single trick (or, occasionally, two). But movement can be put to all kinds of clever tasks (see the next page).And if you watched several proteins each performing its own task but working as a team, you’d begin to appreciate their cumulative “intelligence.”
How does something as seemingly prosaic as DNA’s long, monotonous sequence of only four different nucleotides get converted into the 20,000 or so different kinds of protein molecules that perform daily miracles in our bodies?
That is the business of the cell’s protein-making machinery.
5.1	About Proteins
What Proteins Do
Life’s diversity can be traced to differences in the kinds and arrangements of protein molecules. More than half of the non-water weight of your cells is protein. Proteins do the daily business of living, giving cells their shapes and unique abilities. We’ve alluded to some of proteins’ abilities earlier. Here’s more about the key roles they play.
Enzymes
Enzymes are catalysts — they speed up the breaking apart and putting together of molecules. Their surfaces have special shapes that “recognize” specific molecules, similar to the way a lock accepts only a certain key. Enzymes themselves remain unchanged by the changes they bring about; they can be used over and over again.
Transporters
Special transporter proteins in cell membranes function as tun- nels and pumps, allowing materials to pass in and out of the cell.
Movers
Because the shape of protein chains is mostly determined by weak, easily broken and remade chemical bonds, these chains can shorten, lengthen, and change shape in response to the input or withdrawal of energy. The energy molecule ATP can activate one part of a protein molecule, causing another part of the same molecule to slide or take a “step.” Subsequent removal of ATP causes the protein to return to its original shape. Then the cycle can be repeated.
Supporters
Long chains of folded or coiled proteins can form sheets and tubes — the cell’s equivalent of posts, beams, plywood, cement, and nails.
Regulators
Enzymes that convert one chemical to another must do so in several steps. The first enzyme in a cycle “notices” when enough of the final product builds up and shuts down the assembly line. This ability to respond to feedback is built into the regulator’s structure (see Chapter 6).
Communicators
To work together in harmony, cells must be able to pass messages back and forth. Proteins can act as cells’ chemical messengers. Hormones are examples. Communicator proteins sit on the surface of the receiving cell to gather the incoming signal.
Defenders
Antibodies are proteins with special shapes that recognize and bind to foreign substances, such as bacteria or viruses, surrounding them so that scavenger cells can destroy them and flush them out of the body.
5.2	Multiplying Small Effects
Pumping Iron
Out of the 70,000 or more different kinds of proteins made in human cells, we have selected two — actin and myosin — to show how small molecular events can produce large effects. Actin and myosin are the proteins that make muscle work. Inside muscle cells, actin and myosin genes are translated into many millions of copies of each of these proteins. They line up to form a biochemical ratcheting device that uses ATP for energy to shorten and lengthen itself. This tiny molecular machine leads to the action of a bulging biceps through the simple means of scaling up. Millions of actin-myosin combinations are strung end-to-end in long fibers, and these fibers are bundled together into dense, parallel, elastic cables — the muscle cells. Each microscopic contraction of an actin-myosin combination is amplified into contraction of a cell. Collective cell contractions produce an overall grand contraction — the action of a muscle.
1. Actin molecules are long and thin; myosin molecules are thicker and have many “arms” and “hands” sticking out from their sides. The hands touch the actin molecules.
2. Each unit of contraction consists of two identical arrays of actins attached to discs and facing each other, con- nected by myosin. ATP binds to myosin’s hands, releasing them from actin. The subsequent splitting of ATP to ADP and phosphate causes myosin’s hands to grab actin, and its arms to draw back.
3. The release of ADP and phosphate from myosin causes the arms to make a stroke like an oar, pulling the actins with their attached discs toward each other; this causes contraction.
4. The contraction units are arrayed end-to-end (disc-to-disc) in long fibers called myofibrils. A muscle cell is a cluster of myofibrils.
5. The muscle cells are arranged in many parallel bundles called muscles.
6. Muscles taper into tendons which are attached to the bones they move.
It’s the Same Molecules Everywhere You Look
A WORLD OF SWARMING CHLOROPLASTS I was in a laboratory, using a very expensive microscope. . . . In the circle of light formed by the two eyepieces trained on the translucent leaf . . . I could easily see what I had come to see; the streaming of chloroplasts. . . . Around the inside perimeter of each gigantic cell trailed a continuous loop of these bright green dots. They spun like paramecia; they pulsed, pressed and thronged. A change of focus suddenly revealed the eddying currents of the river of transparent cytoplasm, a sort of “ether” to the chloroplasts, or “space-time,” in which they have their tiny being . . . they swarmed in ever-shifting files around and around the edge of the cell; they wandered, they charged, they milled, raced and ran at the edge of apparent nothingness, the empty-looking inner cell; they flowed and trooped greenly, up against the vegetative wall. Annie Dillard, A Pilgrim at Tinker Creek, 1999
The eddying cytoplasmic currents (described in the observation above) that carry Elodea’s chloro- plasts are driven by exactly the same minuscule actin-myosin motors that make muscles contract and extend, that drive the slime mold’s motion, and that allow white blood cells to engulf invading germs.
Microfilaments composed of the proteins actin and myosin can be found in animal, plant, fungal and bacterial cells. When ATP binds to the myosin molecules in these long fibers, the myosin contacts the actin molecules, causing the microfilaments to contract at the same time and in the same direction. This causes movement of the fluid part of the cell’s contents. Interior organelles, vesicles, and molecules float on these currents like surfers on a wave, traveling rapidly throughout the cell.
Moving materials through a plant’s root cells
In this laser scanning fluorescence micrograph, the actin in actin-myosin microfilaments shows up as bright green fluorescent threads, and each cell’s nuclear DNA fluoresces blue. You can see how the microfilaments permeate the cell, circling the nucle- us and enhancing the intracellular movement of molecules and molecular structures.
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Acetabularia
Acetabularia, whimsically called the mermaids wineglass, is a relatively enormous unicellular alga (it measures from 3 to 10 cm in length, and its “wineglass”capis 1to3cmindiameter).While its linear shape and filmy cap offer a large surface area for the diffusion of materials into and out of the cell, passive diffusion is not enough to move necessary molecules quickly throughout its volume. Cytoplasmic streaming, driven by actin- myosin interactions, is again the solution to this cell’s traffic problem.
A forest of Acetabularia cells.
DOING Science
Allen, Nina S. 1974. Endoplasmic filaments generate the motive force for rotational streaming in Nitella. Journal of Cell Biology 63: 270-287.
This paper describes a clever experiment designed to show that the undulation of micro- filaments in a large algal cell, Nitella, is the cause of the motion of particles throughout the fluid cell interior. Nitella were cultivated and collected. A window was cut into several cells using a mercury arc lamp, which allowed the experimenter to see into the cell and to film cytoplasmic streaming. The movement of particles in the cytosol was filmed by strobe light. The films showed particles moving in a serpentine pattern, which led to the conclusion that they were attached to unseen filaments. When a substance that inhibits actin-myosin interactions but does not affect other molecular structures in the cell was introduced, particle motion stopped. This led to the hypothesis that the filaments were made of actin.
The actin structure in Nitella forms an endless belt that provides enough momentum to sweep the entire cell content in a circle. (Only the actin in the actin-myosin complex flouresces.)
Each amino acid has a different side group with a unique chemical character...
...attached to a back- bone piece that’s the same for every amino acid.
When the backbone pieces are linked together in long chains, they become proteins.
It’s the sequence of the amino acids that distinguishes one protein from another.
5.3	Proteins Are Chains Made from Twenty Amino Acids
Sequence Makes the Difference
Underlying the bewildering variety of protein shapes and sizes is a surprising simplicity. When proteins are unfolded and stretched out, they turn out to be chains of amino acids. The sole determinant of a protein’s natural shape, and consequently its function, is the order of the amino acids in the chain.
There are twenty — and only twenty — amino acids (you can see all of them on page 195). Animals, plants, protists, fungi, and bacteria use some or all of these amino acids in their protein chains. All amino acids contain carbon, hydrogen, oxygen, and nitrogen atoms, and two of them have sulfur atoms as well. Ten of the amino acids have electrically charged side groups that are attracted to water. These cluster on the surface of the folded-up protein chain where it’s easier for them to make contact with the surrounding water in the cell. The other ten amino acids have no electrical charge and so tend to cluster on the inside of the folded-up molecule where they’ll stay dry. The amino acids are linked to each other by strong covalent bonds between their backbone pieces (what we show as chain links). Once a protein is assembled, its covalently linked amino acids form additional weak hydrogen bonds with each other.These easily broken and reformed weak bonds give protein molecules their remarkable ability to change shape, which is the key to their functioning.They also give proteins great flexibil- ity and mobility.
Protein Folding
Proteins find themselves mainly in one of two environments — water or fat. This explains why proteins fold the way they do. A protein in a watery environment folds its fat- liking amino acids tightly inside itself while its water-liking amino acids face the surround- ing water. Proteins that reside in membranes, which are made of fat, do the opposite. Proteins can’t do their work unless they’re folded up correctly.
1. As a protein chain is assembled, it begins to fold, often with the help of small “chaperone” proteins.
2. Usually the fat-liking amino acids turn inward and join together in weak bonds. This forms a stable structure.
3. The water-liking amino acids push to the outside surface where they can do their work.
In its final form, the chain has folded into an intricate shape...
...which we depict this way.
5.4	How Orders Translate into Assembled Boxes of Donuts
Clothespins and Donuts
DoNutArama, a popular donut shop, makes twenty kinds of donuts. The donuts are so good that people buy big boxes of them. And each customer is very particular about having exactly the right kinds of donuts in exactly the right order in the box.
At first, the clerk at the counter tried shouting the orders to the kitchen staff, but they made too many mistakes. Written orders were out because the employees couldn’t read the clerk’s handwriting. Then someone remembered the colored clothespins in the basement. Maybe the clerk could somehow use the clothespins to transmit orders for donuts to the kitchen.
The clothespins came in four colors. The donuts came in twenty varieties.
What’s the most efficient way to use four units to represent twenty units? The clerk worked out a code.
He first tried using combinations of two colors of clothespins: i.e., red + blue = jelly; yellow + red = chocolate; etc. He soon realized that there weren’t enough dif- ferent two-color combinations to represent all twenty donuts. But a three-clothespin code could produce sixty-four (4 × 4 × 4) possible combinations — more than needed for twenty different donuts. So he and his staff worked out and memorized a three-color code: red + blue + yellow = jelly; yellow + red + green = chocolate; etc. As the clerk took the orders, he put the correct color sequence on the line. In the kitchen, the decoder read the code, then hung the proper donut on the hook next to it. The packager took the donuts off the hooks and put them in their proper sequence in the box. Counter orders were transcribed into clothespin sequences and decoded into boxes of donuts, and things worked sweetly ever after.
Four different clothespins, taken three at a time, code for twenty donuts.
Jelly
Coconut
Sprinkles
Custard
Packager
Plain
Maple
Nutty
Banana
Glazed
Chocolate
Blueberry
Marshmallow
Carrot
Carob
Raspberry
Almond
Sugared
Lemon
Pineapple
Prune
5.5	How DNA Information Translates into a Working Protein
Transcription
1. Instructions (Messenger RNA — a copy of a gene)
2. Adaptors (transfer RNA ˆ molecules with amino acids attached)
A transfer RNA is the key “decoding” unit between information and final protein product. Each has a three- letter codon at one end and an amino acid at the other end.
Nucleotides and Amino Acids
A DNA molecule is many, many nucleotides (clothespins) long. It is composed of genes, which are, on the average, some 1200 nucleotides long. Within each gene, the nucleotides are ordered in about 400 groups of three nucleotides apiece. Each nucleotide triplet (called a codon) gets translated into one of the twenty amino acids (donuts). The entire gene will be translated into a protein molecule that is about 400 amino acids long (the packaged donuts).
Here’s how you make a protein. First, copy — transcribe — the sequence of nucleotides in a gene into a single strand of RNA (see Chapter 4, page 174) called messenger RNA (mRNA). Second, attach amino acids to small RNA molecules called transfer RNAs (tRNAs), or adaptors. These act like the decoder with her donut hook. Each adaptor rec- ognizes a particular three-nucleotide codon. Third, bring the adaptors with their attached amino acids and the messenger RNA to a protein synthesis factory called a ribosome (the packager), which links up the amino acids to make the protein.
Four different nucleotides, taken three at a time, code for twenty amino acids.
lysine
asparagine
glycine
proline
arginine
glutamine
alanine
phenylalanine
histidine
serine
valine
methionine
aspartic acid
threonine
leucine
tryptophan
glutamic acid
tyrosine
isoleucine
cysteine
3. Translating machines
Ribosomes are message-reading assembly factories.
A ribosome is where messenger and adaptor pair up. This ensures the correct sequence of amino acids.
4. A finished protein molecule
Here are the four key players in this part of the story: an ATP molecule, an amino acid, an adaptor, and an activating enzyme.
5.6	From DNA to Protein — A Multistep Process
Charging the Adaptor
On the previous pages, we showed the process of transcription of DNA to mRNA in the nucleus, and the translation process in the cell’s cytoplasm by which genes prescribe the order of amino acids in proteins. Now let’s follow the key steps more closely. There has to be a chemical connection between each amino acid and each messenger RNA. Transfer RNA (tRNA) — the adaptor — makes that connec- tion. One end of the adaptor carries a three-nucleotide code.This will match up with three complementary nucleotides on the messenger. A specific enzyme, called an amino acid activating enzyme, energizes each amino acid and then attaches it — just the right one — to the opposite end of the adaptor. Since there are twenty amino acids, there must be at least twenty different activating enzymes and twenty different tRNA adaptors. In the panels on the next page we show the first steps in the con- struction of a protein: energizing amino acids and linking them to their adaptors.
The Basic Idea
An energized amino acid gets put on an adaptor.
The Details
ATP floats near the enzyme and docks in a place tailor-made for it.
Meanwhile, an amino acid floats into a dock nearby.
The two are brought closer together until...
...they bond...
...ejecting two phosphates from ATP.
The amino acid is now energized. (Note how the link is now open.)
Next, the odd-shaped tRNA adaptor floats into view…
...and docks at another nearby site on the enzyme.
The end of the adaptor is brought closer to the amino acid until…
...the two are joined.
Energy flows into the new bond; the “spent” energy molecule is released.
Then the adaptor is released, with its amino acid attached.
5.7	Translation
Assembling the Protein Chain
An energized amino acid has been attached to one end of a tRNA adaptor, which carries at its other end a three-nucleotide code specific for that amino acid. Now the amino acid needs to be linked into a chain with others, in a specific order, to create a specific protein.This next phase requires the help of special machinery that can use the adaptors to “read” the nucleotide triplet codons on the messenger and assemble the appropriate amino acid chains.That’s the job of the ribosomes. A ribosome is made of a larger and a smaller piece, each composed of about equal amounts of ribosomal RNA and protein; it looks a bit like a designer telephone.The ribosome “reads” the tape-like mRNA message three units (one codon) at a time, linking amino acids together as it proceeds.When it gets to the last triplet, which signals “stop,” it releases the finished amino acid chain (the packager closes the donut box).
The Three Key Elements
1. A messenger RNA made in the nucleus (see Chapter 4, page 174).
2. Twenty different tRNAs with one of the twenty different amino acids attached to each of them.
3. A ribosome made of RNA and proteins.
Assembling the Chain
The messenger RNA attaches itself to the smaller subunit of the ribosome.
The first tRNA matches the messen- ger’s first three nucleotides.
The larger subunit joins up with the smaller subunit.
The second tRNA enters a second dock.
The backbone links of the first two amino acids join up.
The messenger shifts to the right, and the first tRNA drops off.
The next tRNA arrives at the second dock to add the next link.
One by one, triplet codons are “read,” and the protein chain grows.
The final triplet codon signals “stop” — no adaptor fits it.
The ribosome separates and drops off the mRNA.
For efficiency in making multiple proteins, messenger RNA is read by more than one ribosome simultaneously.
The solid arrows indicate that protein receives information from DNA via RNA. The broken arrow indicates that, while proteins are needed to transcribe, translate, and replicate DNA, they cannot influence the information in DNA, except through rare copying errors.
5.8	DNA to RNA to Protein
The Flow of Information
The DNA to protein to DNA loop we introduced in Chapter 4 can now be seen more accurately as a DNA to RNA to protein to DNA loop. In a strictly production-line sense, information, in the form of instructions constructed in nucleotide sequences, flows in one direction only: DNA’s message is transcribed into RNA and RNA then gets translated into protein. Proteins are the end of the coded information line — they can’t pass information back to DNA.
In the wider sphere, since it is our proteins, not our DNA, that serve us as our eyes, ears, nose, skin, nerves, etc. — the parts of us that interact with the world we live in — our experiences cannot change the coded sequences in our DNA.This is why the characteristics and behaviors we acquire during our lifetimes cannot be passed on.Whatever happens to our proteins doesn’t change the coded infor- mation in the DNA that made them.
Nevertheless, proteins are keys to the continuity of the loop because they read and translate DNA’s instructions during an organism’s lifetime and are essential to copying DNA so that it can be passed to the next generation.And proteins con- trol which parts of DNA’s instructions — which genes — are to be expressed; i.e., they turn genes on and off based on information from their surroundings.
Finally, proteins do affect the information in DNA in an evolutionary sense. A substantial number of mutations occur in copying errors, so that information is sometimes altered as it is passed along. In these ways, proteins influence the flow of all information in living systems.
RNA’s four nucleotides
AUCG
Backbone: ribose and phosphate
DNA’s four nucleotides
ATCG
Backbone: deoxyribose and phosphate

5.9	Key Discovery
“The conclusion was inescapable...”
Polyphenylalanine
Polyuracil
 “a triplet of U’s = phenylalanine”
Cracking the Genetic Code
In 1961, Marshall Nirenberg and Johann Matthaei, two young biochemists at the National Cancer Institute in Bethesda, Maryland, made an astonishing discovery. Not yet aware of the discovery of messenger RNA in Britain and France, they were searching for something like it: evidence that some type of RNA might program ribosomes to make protein. They took any samples of RNA they could lay their hands on and incubated them with ribosomes from bacteria, along with activating enzymes, ATP, transfer RNAs, and a mixture of amino acids. They looked to see if any of the RNAs simulated protein synthesis. The results were not particularly encouraging until, by chance, they added an artificial RNA — polyuridylic acid (UU-U-U...) — chains made of one nucleotide, uracil, linked to each other as in natural RNA. Incredibly, the ribosomes obediently “read” the “poly U” chains into an artificial “protein,” polyphenylalanine — long chains of the single amino acid phenylalanine! The conclusion was inescapable: The triplet code for phenylalanine must be UUU.
The exciting wider implication: If ribosomes can be induced to translate RNAs of any nucleotide sequence into protein, then RNAs of known nucleotide sequence could be incubated with ribosomes and watched to see what kind of amino acid sequence came out. Here lay the solution to the genetic code! Nirenberg and Matthaei pounced, as did others who learned of their discovery. A frenzy of experimentation ensued, with the result that all sixty-one of the triplet codes for the twenty amino acids were identified by 1965.
How to Read the Genetic Code
The chart on the right summarizes the genetic code. Read it like a map with coordinates. Three nucleotides code for one amino acid. Each triplet of nucleotides is called a codon. If you want to find the amino acid whose code is CAU, for example, find the box where C in the left-hand column meets A in the top row. This box contains histidine and glutamine. From this box look across to the right-hand column and find U. So histidine is represented by CAU. Note that it’s also represented by CAC.
Life has used all but three of the sixty-four possible codons that can be made using four nucleotides. So most of the amino acids are represented by more than one triplet. The three triplets that don’t code for an amino acid instead signal the protein-making machinery to stop; they are called stop codons.
5.10	The Unity of Biology
A Light That Blinked...
Life first strikes us with its diversity. Evolution has filled every niche: Bacteria thrive in hot springs; fish plumb the depths of the sea; birds soar skyward, defying gravity. But when we look below the surface at the ways molecules work in cells, we cannot but marvel at their unity. All living creatures use DNA and RNA to store and replicate information, building them from the same four nucleotides. They make those nucleotides using very similar pathways. They translate nucleotide chains into proteins using the same twenty amino acids and the same genetic code. They use very similar translation apparatus — ribosomes, tRNAs, mRNAs, activating enzymes. If we take ribosomes from bacteria and put them in a test tube, they’ll translate human messenger RNAs into human proteins — and vice versa. And many of the proteins — supporters, movers, communicators, transporters, and catalyzers — when dissected into their primary amino acid sequences — are quite similar in most creatures throughout the living world.
The realization dawns on us that we all had common beginnings. Billions of years ago, a tiny light blinked on somewhere and has come to illuminate every nook and cranny of our Earth’s surface.
“We are educated to be amazed by the infinite variety of life forms in nature; we are, I believe, only at the beginning of being flabbergasted by its unity.” Lewis Thomas
Some of the Things You Learned About in Chapter 5
actin-myosin interactions 186–189 activating enzymes 184 adaptors 196 amino acid side groups 190, 195 common beginnings 204 communicators 185
cytoplasmic streaming 188–189 defenders 185 enzymes 184 messenger RNA (mRNA) 194 movers 184
protein folding 191 proteins 190,201 regulators 185 ribosomes 195,198 sequence 190 supporters 185 transcription 194,200 transfer RNA (tRNA) 194 translation 198,200 transporters 184
Questions About the Ideas in Chapter 5
1.	Name at least four of the key activities of proteins that make life possible.
2.	What two things have to be done to amino acids before they can be linked together in a protein molecule?
3.	Why is transcription necessary? Why don’t cells make their proteins directly from DNA?
4.	What are the key elements of the translation machinery?
5.	How would you go about testing that a bacterium’s protein-making machinery could make a human protein?
6.	What is a triplet and what is its function?
7.	How would each of the following mutations affect the final sequence of a specific protein (or even its production)?
(a) a change in the DNA sequence for the protein itself (b) a mutation in the gene for one of the specific activating enzymes (c) a change in the transfer RNA sequence
8.	How is a regulatory protein like an old-fashioned spring clothespin? What other parallels can you suggest that might evoke the way proteins change shape and the work they do?
9.	Most mutations are harmless and a very few are even beneficial to organisms. How is this possible if the usual result of a mutation is to change the amino acid sequence of a protein and affect its correct folding? (Hint: Remember that there are 20 amino acids and 64 possible combinations of triplets using the four DNA bases.)
10.	Protein synthesis is a complicated and dynamic process with numerous steps, many of which require ATP for the energy to move molecules or to form new bonds. Can you think of a reason why so many steps are required?
References and Great Reading
Allen, N. S. 1974. Endoplasmic filaments generate the motive force for rotational streaming in Nitella. Journal of Cell Biology 63: 270-287.
Bronowski, J. 1990. Science and Human Values. New York: HarperCollins.
Gunning, B. E. S. and M.W. Steer, 1996. Plant Cell Biology: Structure and Function. Sudbury, MA: Jones and Bartlett Publishers.
Hartl, D. L. and E.W. Jones, 1999. Essential Genetics, 2E. Sudbury, MA: Jones and Bartlett Publishers. Hoagland, M. 1990. Toward the Habit ofTruth:A Life in Science. NewYork:W.W. Norton. Strickberger. M. 2000. Evolution, 3E. Sudbury, MA: Jones and Bartlett Publishers.
Williamson, R.E. 1980. Actin in motile and other processes in plant cells. Canadian Journal of Botany 58: 766-772.
WEB Connection
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