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Trends in Biotechnology

150319 TB 6 The cell as a basic unit of a living organism each carrying out

many different functions

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When we look at cells we now also look at subcellular organization and components. We have to understand what the macromolecules in cells are, where they are found and how they work.

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Here is a very brief review of biochemistry and how this affects cells.

The biochemistry is grouped into functions.And the functions are related to the organelles of the cell.

The following material is based and borrowed from:Biochemistry online: An approach based on chemical logic – this is the rectangle diagram of the cellhttp://employees.csbsju.edu/hjakubowski/classes/ch331/bcintro/default.htmland Inside the Cell – these include the other diagrams except where stated. http://publications.nigms.nih.gov/insidethecell/pdf/inside_the_cell.pdfWith other material from Inside the Cell NIH Publication No. 05 1051 Revised September 2005 http://www.nigms.nih.gov

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Three important ideas from chemistry are:

The structure, charge, and size of a molecule will cause the function/activity of the molecule;

Binding reactions are what start all biological events; and

Chemical ideas can be applied to the behavior of macromolecules.

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This leads us to some questions. First we will think of a general cell.

6http://en.wikipedia.org/wiki/Cell_(biology)#/media/File:Animal_cell_structure_en.svg artist is LadyofHats (Mariana Ruiz)

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ACTUAL SIZE (AVERAGE)SIZE WHEN

MAGNIFIED 3 MILLION TIMES (meters)

Cell diameter 30 micrometers*

90Nucleus diameter 5 micrometers

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Mitochondrion lengthTypically 1–2 micrometers, but can be up to 7 micrometers long

3 - 7Lysosome diameter 50–3,000 nanometers

0.15 - 9Ribosome diameter 20–30 nanometers

0.06 - 0.09Microtubule width 25 nanometers

0.75Intermediate filament

width10 nanometers

0.03Actin filament width 5–9 nanometers

0.015 - 0.027Cell membrane 6 or more nanometers

0.018

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We can now redraw this cell by thinking about the different functions of the cell.

http://employees.csbsju.edu/hjakubowski/classes/ch331/bcintro/default.html

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This diagram shows grouping of functions• Storage and retrieval of information• Enclosing structures and separating the cell from the

environment.• Making, folding and transporting proteins• Making membrane receptors and channels, and letting

things through the membrane.• Differentiation and gene control• Regulation and linking of biochemical reactions• Catalysis of biochemical reactions• Sensing and responding to the environment and

sending signals

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Storage and retrieval of informationGenetic information is stored in DNA

which can be replicated to make copies of that information.

DNA information is transcribed into an RNA molecule.

This information is then translated into a protein.

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Genetic material - DNA – is in the nucleus.

Genes, each with a set of helper molecules

DNA contains information for proteins.

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DNA ligase I repairing chromosomal damage. The three visable protein structures are:• The DNA binding domain (DBD) which

is bound to the DNA minor groove both upstream and downstream of the damaged area.

• The OB-fold domain (OBD) unwinds the DNA slightly over a span of six base pairs and is generally involved in nucleic acid binding.

• The Adenylation domain (AdD) contains enzymatically active residues that join the broken nucleotides together by catalyzing the formation of a phosphodiester bond between a phosphate and hydroxyl group.

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Nucleus is surrounded by two membranes, together known as the nuclear envelope.

The nuclear envelope has many octagonal pores.

Nuclear pores allow chemical messages to exit and enter the nucleus.

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Each cell contains about 10 billion protein molecules of approximately 10,000 different varieties.

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Proteins are responsible for many tasks, eg: Carrying oxygen in blood (hemoglobin), Digesting food (eg. amylase, pepsin, and lactase), Defence from invading microorganisms (antibodies), and Speeding up chemical reactions (enzymes). Specially designed proteins give elasticity to skin (elastin) and strength to hair and fingernails (keratin).

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Protein production starts in the nucleus. Genes, which are made of DNA, contain the instructions for making proteins. Many other factors eg.

•Diet, •Activity level, and •Environment

can affect when and how the body will use these genes.

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1st step in building proteins is reading the genetic code contained in DNA. This is called transcription.Inside the cell nucleus, where DNA is packaged in chromosomes, are RNA polymerases. Made of 12 different small proteins, these molecular machines first pull apart the two strands of DNA, then transcribe the DNA into RNA.

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Researchers have used X-ray crystallography to help find how transcription occurs. Eg, Roger Kornberg used this tool to obtain a detailed, three- dimensional image of RNA polymerase. This suggests that the RNA polymerase enzyme uses a pair of jaws to grip DNA, a clamp to hold it in place, a pore through which RNA components enter, and grooves for the completed RNA strand to thread out of the enzyme.

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You can see Professor Kornberg’s slides at http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2006/kornberg-slides.pdf

Pp 41 on

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Helper molecules may then cut and fuse together pieces of RNA and make a few chemical modifications to give the finished products - correctly sized and processed strands of messenger RNA (mRNA). Completed mRNA molecules carry genetic messages to the cytoplasm, where they are used as instructions to make proteins.

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Specialized proteins and small RNA molecules escort the mRNA out of the nucleus through pores in the nuclear envelope.

A sequence of chemical reactions that use ATP drives this export process.

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Once in the cell’s cytoplasm, each mRNA molecule serves as a template to make a single type of protein. A single mRNA message can be used over and over again to create thousands of identical proteins.

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This process, called translation, is carried out by ribosomes, which move along the mRNA and follow its instructions. The mRNA instructions are a string of units that, in groups of three, code for specific protein building blocks called amino acids. Ribosomes read the mRNA units in sequence and string together the corresponding amino acids in the proper order.

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Ribosomes - molecular machines made up of more than 70 proteins and 4 strands of RNA.

Ribosomes assemble all the cell’s proteins. Newly made protein chains come out of

ribosomes, and thread directly into the ER (Endoplasmic reticulum).

Enzymes add special sugars to the protein.

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In 1999 scientists got the first structural picture of an entire ribosome.

This image shows a bacterial ribosome making a protein.

Ribosomes are made mostly of RNA.

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Knowledge of ribosomal structures could lead to improved antibiotic medicines.

All cellular organisms, including bacteria, have ribosomes.

But the precise shapes of ribosomes differ in several specific ways between humans and bacteria.

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Several antibiotic medicines currently on the market work by inhibiting the ribosomes of bacteria.

Many microorganisms have developed resistance to these medicines.

Need new antibiotics to replace those that are no longer effective in fighting disease.

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Imaging techniques like X-ray crystallography have produced molecular pictures of antibiotics grabbing onto a bacterial ribosome.

These 3D images gives new ideas about how to design molecules that grip bacterial ribosomes even more strongly.

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This may result in new and more effective antibiotic drugs.

Therapies that knock out bacterial ribosomes (kills the bacteria) should work without affecting the human hosts.

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Molecules called transfer RNAs (tRNAs) that bring amino acids from the cytosol to the ribosome. One end of the L-shaped tRNA matches up with a three-unit mRNA sequence while the other end carries the appropriate amino acid.

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One at a time, the tRNAs clip onto the mRNA in a cavern deep within the ribosome, allowing the ribosome to stitch together the amino acids in the right order. A finished amino acid chain can range in length from a few dozen to several thousand amino acids. Some proteins are made up of only one amino acid chain. Others, especially large proteins, contain two or more chains.

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Translation consumes lots of energy, but it happens very fast. In bacteria, for example, ribosomes can join together 20 amino acids in 1 second.

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Some three-unit sequences in the mRNA message can immediately halt protein production. Reading one of these mRNA stop signs indicates to the ribosome that the new protein has all the amino acids it needs, and translation ends.

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At this point, most proteins made by free-floating ribosomes are essentially complete. They will remain in the cytosol, where they conduct business—such as passing chemical messages in the cell.

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Proteins made by ribosomes on the rough ER. Inside the rough ER, enzymes add specialized chains of sugar molecules (carbohydrates) to proteins in a process called glycosylation.

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Next, the proteins traverse the Golgi, where the sugar groups may be trimmed or modified in other ways to create the final protein.

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Unlike genes and proteins, carbohydrates are not based on a genetic template. As a result, they are more difficult to study because researchers cannot easily determine the sequence or arrangement of their components. Scientists are only just beginning to learn about the critical roles carbohydrates play in many life processes.

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For example, without the carbohydrates on its outer surface, a fertilized egg would never implant into a woman’s uterus, meaning it would never develop into a baby. Also, without sticky sugar molecules to slow down immune cells, they would not stop to help fight infection.

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Sugars attached to lipids on the surface of red blood cells define a person’s blood type (A, B, AB, or O). Carbohydrates even help proteins fold up into their proper shape and dictate where proteins go and which other molecules they can interact with.

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Some children are born who can’t glycosylate their proteins, a disorder called carbohydrate deficiency glycoprotein syndrome.

This disease affects virtually every part of the body, causing symptoms like mental retardation, neurological defects, and digestive problems.

Glycosylation, then, does more than just add a sugar coating. It’s an essential process that gets proteins ready for action.

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Enclosing structures and separating the cell from the environment

Lipids join thermodynamically spontaneously to form structures like biological membranes.

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Without membranes cells and life could not exist.

Cell membrane - made up of proteins and lipids. Membrane’s location and role in the body - lipids can make up anywhere from 20 to 80 percent of the membrane, - the remainder is proteins. Cholesterol is a type of lipid that helps stiffen the membrane.

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About half of all human proteins include chains of sugar molecules that are critical for the proteins to function properly.

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Making, folding and transporting proteins

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Proteins fold to form structures with a unique 3D shape.

These 3D structures give a specific function to proteins.

Proteins are synthesized in the cytoplasm and transported to where they are needed

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The Golgi complex, also called the Golgi apparatus or the Golgi.

• receives newly made proteins and lipids from the ER,

• finishes the production• add a molecular address • sends them to their final

destinations.

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Cytoskeleton gives cells –shape, –strength, –the ability to move–constantly shrink and grow to meet

the needs of the cell

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Three fibers of the cytoskeleton – • microtubules,• intermediate filaments, and • actin filaments.

Each type of fiber looks, feels, and functions differently.

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Microtubules•Made of tubulin•separating duplicate chromosomes

when cells copy themselves •act as structures on which

molecules and materials move. •hold the ER and Golgi in stacks

• form the main component of flagella and cilia.

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Intermediate filaments - vary greatly according to their location and function in the body.

• Some form tough coverings, such as in nails, hair, and the outer layer of skin

• Others are in nerve cells, muscle cells, the heart, and internal organs.

In each of these tissues, the filaments are made of different proteins. So if doctors analyze intermediate filaments in tumors, they can determine the origin of —and possible treatments for—some kinds of cancer.

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Actin filaments • Two chains of the protein actin twisted

together. • Can gather together into bundles, weblike

networks, or even three-dimensional gels. • Shorten or lengthen to allow cells to move

and change shape. • Together with myosin, actin filaments make

possible the muscle contractions.

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To reach its destination, a newly created protein must be moved through the cytosol, moving past obstacles, such as organelles, cytoskeletal fibers, and many molecules. Well-organized systems bring proteins to the places where they are needed.

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A protein called kinesin (blue in the image above) is in charge of moving cargo around inside cells and helping them divide. It's powered by biological fuel called ATP (bright yellow) as it scoots along tube-like cellular tracks called microtubules (gray).

http://publications.nigms.nih.gov/biobeat/gallery/kinesin.htmlCourtesy of biochemist Charles Sindelar, Brandeis University

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Vesicles

Membranes present an important obstacle. The cell’s cytosol, the insides of organelles, and many proteins are water-soluble.But the insides of membranes are fat-soluble (oily). Oil and water don’t mix.

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Water-loving (hydrophilic) proteins are wrapped in vesicles.They can cross the fatty membranes surrounding lysosomes, the ER, or the Golgi.Vesicles are protective membrane bubbles.

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Eg. Proteins from the ER to the Golgi.

A small portion of the ER membrane pinches off, enveloping proteins in a vesicle that has a special molecular coat. This vesicle then travels to the Golgi.

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Docking sites on the Golgi permit vesicles to attach to and fuse with its outer membrane to release their contents inside. The same process takes proteins in vesicles from the Golgi to lysosomes or to the cell’s surface.

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Cells also use vesicles to carry nutrients and other materials into the cell in a process called endocytosis. White blood cells use endocytosis to fight infection.

They engulf bacteria in large vesicles. The vesicles then fuse with lysosomes, which break down the bacteria into molecular pieces the cell can use.

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Endocytosis occurs continuously, and cells use their entire membrane every 30 minutes. Exocytosis, counterbalances endocytosis. Cells use this process to put wastes out of the cell and to replace membrane.

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Secretory vesicles James Rothman and Randy Schekman:

cells use secretory vesicles to organize their activities and communicate with their environment.

They used genetically altered yeast cells to study cell secretion. They discovered how cells use vesicles to direct proteins and other molecules to their proper destinations.

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This research taught scientists that vesicles are very important for cells. Vesicle transport underlies many processes, such as the secretion of insulin to control blood sugar, nerve cell communication, and the proper development of organs.

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The work also helped scientists learn to use yeast cells as protein factories. Genetically altered yeast cells now pump out many important products, including approximately one-quarter of the world’s insulin supply and a key ingredient in hepatitis B vaccines.

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Molecular Motors

Vesicles move with direction. Like many other materials inside the cell, including some organelles, they often are carried by small molecular motors along tracks formed by the cytoskeleton.

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Motors are used in the cell to get many things done —• copying DNA (and fixing it when a

mistake is made),• making ATP and proteins, and • putting molecules in the correct places

during development to make sure the body is assembled correctly.

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In recent years, scientists have discovered that the workings of every motor they examined use the same two ingredients: • an energy source (usually ATP) and • chemical reactions.

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Making membrane receptors and channels, and letting things

through the membrane.

• Proteins form membrane receptors and channels

• Proteins allow passage of hydrophilic molecules into and out of the cell.

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Cells transport smaller molecules, like water and charged particles (ions), across membranes. These molecules travel through hollow or gated proteins that form channels through membranes.

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Channel proteins are one family of proteins that function within the cell’s surface membrane.

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Channel proteins transport ions like sodium and potassium that are critical to many biological processes, such as the beating of the heart, nerve impulses, digestion, and insulin release. Unfortunately, channel proteins are difficult to study because they cannot easily be isolated from the membrane in either their natural or active states.

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Differentiation and gene control

Proteins catalyze and regulate how specific proteins are made.A cell knows what kind of cell it is to be. A cell knows what kind of proteins to make.

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Cells are different

Liver cells are not like nerve cells. Muscle cells are different to white blood cells. Yet every cell (with just a few exceptions) is enclosed in a membrane, contains a nucleus full of genes, and has ribosomes, mitochondria, ER, and Golgi.

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How can cells be so similar, yet so different? Despite many years of hard work, cell biologists still don’t fully understand how developing cells turn into all the different types in your body. But, they do know that this process, called differentiation, is governed by genes.

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The body controls the genes of each cell type differently.

Depending on where in the body it is located, a given gene can be turned off, weakly on, or strongly on.

For example, the gene for globin, which makes hemoglobin, is strongly on in cells that will mature into red blood cells and off in every other cell type.

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Cells control the expression of genes by controlling RNA polymerase.

For genes that are strongly on, cells use special molecular tags to bring in RNA polymerase and to ensure that the machine works well transcribing those genes.

For genes that are off, cells use different tags to repel RNA polymerase.

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Embryonic stem cells have gene expression so wide that it has unlimited potential to become any kind of cell in the body.

These undifferentiated cells cease to exist a few days after conception.

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Regulation and linking of biochemical reactions

Enzymatic reactions be regulated so the cell can biosynthesize and get energy.

Reactions are brought together in pathways.

Whole pathways are regulated.

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Through a series of chemical reactions, mitochondria transfer energy in small packets from glucose into ATP.

All that’s left are carbon dioxide and water, which are discarded as wastes.

This process is extremely efficient. Cells convert nearly 50 percent of the

energy stored in glucose into ATP.

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There are many reactions in the cell and many are linked to form biochemical pathways.

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The reactions are controlled in many ways. The cell is obviously a very complex system. Scientists interested in metabolomics study

how metabolism (the body’s breakdown of certain molecules and the synthesis of others) is governed by thousands of enzymes and signaling networks in an organism.

-omics tagged on to the end of a word means a systematic survey of an entire class of molecules.

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Catalysis of biochemical reactions

Enzymes catalyze biochemical reactions.

We can find out how these work.

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Sensing and responding to the environment and sending signals

Cells sense environmental signals and respond the correct way.

Cells send out chemical signals.

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Following is a diagram which shows the different proteins of cells (different genes) grouped by function.

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Write down these groups from the biggest to the smallest in the following table.

Write down the functions of each group.Group Functions of Group