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PROTEIN STRUCTURE Amino Acid Monomers Amino acids are the monomers of proteins There are 20 different types of amino acids Amino acids differ in their properties due to differing side chains, called R groups When you see an ‘R’ on a molecule, it means that multiple different things could be in the spot that says ‘R.’ There is no element with the symbol R. Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings Fig. 5-UN1 Different things will be in the R spot depending on which of the 20 amino acids you are looking at. Amino group Carboxyl group Fig. 5-17 Glycine (Gly or G) Nonpolar Valine (Val or V) Alanine (Ala or A) Methionine (Met or M) Leucine (Leu or L) Trypotphan (Trp or W) Phenylalanine (Phe or F) Isoleucine (Ile or I) Proline (Pro or P) Polar Serine (Ser or S) Threonine (Thr or T) Cysteine Tyrosine (Cys or C) (Tyr or Y) Asparagine Glutamine (Asn or N) (Gln or Q) Electrically charged Acidic Aspartic acid Glutamic acid (Glu or E) (Asp or D) Basic Lysine (Lys or K) Arginine (Arg or R) Histidine (His or H) Chart showing the 20 different amino acids. The part in the white area is the R group (different in each amino acid). The part on the bottom (gray area) is the same for all amino acids. If you can recognize that part, you’ll be able to tell if something is an amino acid or not. Polypeptides Polypeptides are polymers built from amino acids A protein consists of one or more polypeptides Polypeptides range in length from a few to more than a thousand monomers Each polypeptide has its amino acids in a different order. Some polypeptides have more of some kinds of amino acids, others include different kinds of amino acids. Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings Fig. 5-18 Peptide bond Amino acids forming a polypeptide by a dehydration reaction (a) Side chains Peptide bond Backbone (b) Amino end (N-terminus) Carboxyl end (C-terminus) Protein Structure and Function A functional protein consists of one or more polypeptides twisted, folded, and coiled into a unique shape Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings Antibody protein Protein from flu virus The sequence of amino acids determines a protein’s three-dimensional structure A protein’s structure determines its function Sickle-Cell Disease: A Change in Protein Structure A slight change in protein structure can affect a protein’s structure and ability to function Sickle-cell disease, an inherited blood disorder, results from a single wrong amino acid in the protein hemoglobin The DNA gives instructions about which amino acids to put together to make a protein. If there’s a mistake in the DNA (called a mutation), you can get a protein with a wrong amino acid; the mutated protein may not function well Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings Fig. 5-22c 10 µm Normal red blood cells are full of individual hemoglobin molecules, each carrying oxygen. 10 µm Fibers of abnormal hemoglobin deform red blood cell into sickle shape. What Determines Protein Structure? In addition to the sequence of amino acids, physical and chemical conditions can affect protein shape Alterations in pH, salt concentration, temperature, or other environmental factors can cause a protein to unravel This loss of a protein’s original shape is called denaturation A denatured protein is biologically inactive (one reason doctors worry about very high fevers) Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings Fig. 5-23 Denaturation Normal protein Renaturation Denatured protein Functions of Proteins: Lots! Structure: -Proteins are embedded in cell membranes with phospholipids -Proteins direct DNA to fold into chromosomes before cell division Image from: http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/chromosome.gif Proteins control gene expression by turning genes on and off http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/protsyn/regulation/ionoind.html Images from: http://www.imac.auckland.ac.nz/vaccines/vacc_graph.htm Proteins fight germs. Antibodies are proteins. ANTIBODIES ATTACK & KILL THEM Proteins help with transport. Proteins in cell membranes move molecules in and out of cells. Image from: http://bio.winona.msus.edu/berg/ANIMTNS/FacDiff.htm Proteins help with transport. Hemoglobin in blood helps transport oxygen all over the body. Image from: http://www.cellsalive.com/pics/cover4.gif Some proteins act as hormones. Eating carbohydrates puts glucose in your bloodstream. Insulin is a protein hormone that controls blood glucose. Image from: http://www.cibike.org/CartoonEating.gif Insulin function image by Riedell using Glycogen image modified from: http://www.msu.edu/course/lbs/145/smith/s02/graphics/campbell_5.6.gif People with diabetes can’t make enough insulin, so glucose stays in their blood instead of being stored by cells. Shots can replace the insulin and help to reduce blood sugar. Image modified from: http://sonya.lanecurrent.net/Health/Images/meds.gif Fig. 5-16 Substrate (sucrose) Glucose OH Fructose HO Enzyme (sucrase) H2O PROTEIN SYNTHESIS The Flow of Genetic Information Your DNA is a set of instructions that your cells follow to make proteins. The proteins you have determine your traits. Gene expression, the process by which DNA directs protein synthesis, has two stages: transcription and translation Basic process: DNA mRNA Protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-3a-1 TRANSCRIPTION DNA mRNA (a) Bacterial cell Fig. 17-3a-2 TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell Fig. 17-3b-1 Nuclear envelope TRANSCRIPTION DNA Pre-mRNA (b) Eukaryotic cell Fig. 17-3b-2 Nuclear envelope TRANSCRIPTION RNA PROCESSING mRNA (b) Eukaryotic cell DNA Pre-mRNA Fig. 17-3b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell Summary of Transcription and Translation Transcription DNA mRNA (happens in the nucleus) Translation mRNA polypeptide (happens in the cytoplasm) - Ribosomes help The Genetic Code How does DNA tell us which kinds of amino acids to put together for each protein? There are 20 amino acids, but there are only four nitrogenous bases in DNA How many bases correspond to an amino acid? Remember – Proteins are strings of amino acid monomers put together Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Codons: Triplets of Bases Every 3 DNA nucleotides represent a triplet, which your ribosomes read like a word. Each 3-letter “word” on the DNA codes for a specific amino acid Example: AGT on the DNA tells the ribosome to add the amino acid Serine Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings During transcription, mRNA nucleotides attach to one of the two DNA strands (called the template strand). http://www.phschool.com/science/biology_place/biocoach/transcription/tcproc.html During translation, the mRNA 3-letter words, called codons, are read in the 5 to 3 direction (the letter at the 5’ end is at the beginning of the codon word). Each codon specifies which amino acid should be added to the polypeptide next DNA has triplets, which attach to mRNA codons Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-4 DNA molecule Gene 2 Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid Cracking the Code All 64 codons were deciphered by the mid-1960s Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation Just as some words mean the same thing, multiple codons can give the same amino acid Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Reading frames For the sequence ‘AUGCCGAC’, you could start at the beginning and read the codons “AUG, CCG” If you used a different reading frame, you could read “UGC, CGA” or “GCC, GAC” If you start in the wrong reading frame, you’ll combine the wrong amino acids to make the wrong protein. Since AUG is the start codon, ribosomes look for an AUG and start reading there. Third mRNA base (3 end of codon) First mRNA base (5 end of codon) Fig. 17-5 Second mRNA base Evolution of the Genetic Code The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transplanted from one species to another The same sequence of nitrogenous bases in the DNA will code for the same amino acids and proteins in almost all species. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-6 (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene http://projecthdesign.com/wp-content/uploads/2007/11/landmineflowers1.jpg http://www.bbc.co.uk/news/scienceenvironment-14882008 http://en.wikipedia.org/wiki/File:GloFish.jpg http://www.wired.com/science/plan etearth/news/2008/01/gm_insects# Details of Transcription RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine RNA has U instead of T Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-7b Nontemplate strand of DNA Elongation RNA polymerase 3 RNA nucleotides 3 end 5 5 Direction of transcription (“downstream”) Newly made RNA Template strand of DNA Fig. 17-8 1 Promoter A eukaryotic promoter includes a TATA box Template 5 3 3 5 TATA box Start point Template DNA strand 2 Transcription factors Several transcription factors must bind to the DNA before RNA polymerase II can do so. 5 3 3 5 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex The DNA sequence where RNA polymerase first attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DNA that is transcribed is called a transcription unit Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings RNA Polymerase Binding and Initiation of Transcription Before transcription can start, Transcription factors attach to the promoter region and help the RNA polymerase bind onto the DNA in the right place A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Elongation of the RNA Strand As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes A gene can be transcribed by several RNA polymerases at a time Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-7a-2 Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 Unwound DNA 3 5 RNA transcript Template strand of DNA Fig. 17-7b Nontemplate strand of DNA Elongation RNA polymerase 3 RNA nucleotides 3 end 5 5 Direction of transcription (“downstream”) Newly made RNA Template strand of DNA Fig. 17-7a-3 Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 5 RNA transcript 3 5 Fig. 17-7a-4 Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 5 3 5 RNA transcript 3 Termination 5 3 3 5 5 Completed RNA transcript 3 Animation: http://www.dnalc.org/resources/3d/12-transcription-basic.html Fig. 17-3b-3 Eukaryotic cells process RNA During RNA processing, both ends of the primary transcript are usually altered Also, usually some interior parts of the molecule are cut out, and the other parts spliced together Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way: The 5 end receives a modified nucleotide 5 cap The 3 end gets a poly-A tail Why add these ends? They seem to make it easier to move mRNA out of the nucleus They protect mRNA from hydrolytic (causing hydrolysis) enzymes They help ribosomes attach to the 5 end Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-9 Protein-coding segment PolyA signal 5 G 3 P P P 5 Cap AAUAAA 5 UTR Start codon Stop codon 3 UTR AAA…AAA Poly-A tail Exons, Introns, and RNA Splicing • • • • Most eukaryotic genes and their RNA transcripts have pieces that don’t code for useful proteins. These noncoding regions are called introns The other parts are called exons because they are eventually expressed (translated into amino acid sequences) RNA splicing removes the introns and joins the exons together, creating mRNA with a continuous coding sequence Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-10 5 Exon Intron Exon Exon Intron 3 Pre-mRNA 5 Cap Poly-A tail 1 31 30 Coding segment 104 105 146 Introns cut out and exons spliced together mRNA 5 Cap Poly-A tail 1 146 The Functional and Evolutionary Importance of Introns Some genes can code for more than one kind of protein, depending on which segments are treated as exons during RNA splicing These variations are called alternative RNA splicing Because of alternative RNA splicing, the number of different proteins an organism can produce is much greater than its number of genes http://www.dnalc.org/view/16941-2D-Animation-of-Alternative-RNA-Splicing.html http://www.dnalc.org/view/16940-Alternative-RNA-Splicing.html Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings After RNA processing, the mRNA is translated. A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) Molecules of tRNA are not identical: Each carries a specific amino acid on one end Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-13 Amino acids Polypeptide tRNA with amino acid attached Ribosome tRNA Anticodon Codons 5 mRNA 3 The Structure and Function of Transfer RNA A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-14a 3 Amino acid attachment site 5 Hydrogen bonds Anticodon (a) Two-dimensional structure Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule tRNA is roughly L-shaped Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-14b Amino acid attachment site 5 3 Hydrogen bonds 3 Anticodon (b) Three-dimensional structure 5 Anticodon (c) Symbol used in this book Accurate translation requires two steps: First: a correct match between a tRNA and an amino acid (tRNA with an amino acid attached is “charged” tRNA) Second: a correct match between the tRNA anticodon and an mRNA codon Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Ribosomes Ribosomes help tRNA anticodons to pair up with specific mRNA codons in protein synthesis The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-16b P site (Protein holding site) E site (Exit site) A site (Arrival site) E P A mRNA binding site Large subunit Small subunit A ribosome has three binding sites for tRNA: The A site holds the new tRNA that carries the next amino acid that will be added The P site holds the tRNA that carries the growing polypeptide chain The E site is the exit site, where discharged tRNAs leave the ribosome Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Initiation of Translation First, a small ribosomal subunit binds with mRNA and a special initiator tRNA Then the small subunit moves along the mRNA until it reaches the start codon (AUG) Proteins called initiation factors bring in the large subunit The initiator tRNA sticks to the AUG codon. What is the anticodon on this tRNA? Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-17 3 U A C 5 5 A U G 3 Initiator tRNA Large ribosomal subunit P site GTP GDP E mRNA 5 Start codon mRNA binding site 3 Small ribosomal subunit 5 A 3 Translation initiation complex Elongation of the Polypeptide Chain A tRNA recognizes the mRNA codon its anticodon fits with and binds to the mRNA in the A site The tRNA in the P site passes the chain of amino acids to the tRNA in the A site Both tRNAs move over to the next site The tRNA that is now in the E site exits and the process repeats Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-18-1 Amino end of polypeptide E 3 mRNA 5 P A site site Fig. 17-18-2 Amino end of polypeptide E 3 mRNA 5 P A site site GTP GDP E P A Fig. 17-18-3 Amino end of polypeptide E 3 mRNA 5 P A site site GTP GDP E P A E P A Fig. 17-18-4 Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P A site site 5 GTP GDP E E P A P A GDP GTP E P A Termination of Translation Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome The A site accepts a protein called a release factor The release factor adds a water molecule instead of an amino acid The polypeptide, the mRNA, and the two ribosomal subunits come apart and float off into the cytoplasm Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-19-1 Release factor 3 5 Stop codon (UAG, UAA, or UGA) Fig. 17-19-2 Release factor Free polypeptide 3 5 5 Stop codon (UAG, UAA, or UGA) 3 2 GTP 2 GDP Fig. 17-19-3 Release factor Free polypeptide 5 3 5 5 Stop codon (UAG, UAA, or UGA) 3 2 GTP 2 GDP 3 Polyribosomes A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome Polyribosomes enable a cell to make many copies of a polypeptide very quickly Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-20 Growing polypeptides Completed polypeptide Incoming ribosomal subunits Start of mRNA (5 end) (a) End of mRNA (3 end) Ribosomes mRNA (b) 0.1 µm Targeting Polypeptides to Specific Locations Cells have 2 kinds of ribosomes: free ribosomes and bound ribosomes Free ribosomes mostly synthesize proteins that are used in the cytoplasm Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell Ribosomes are identical and can switch from free to bound Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings • • • • Protein synthesis always begins in the cytoplasm It finishes in the cytoplasm unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER are marked by a signal peptide A signal-recognition particle (SRP) binds to the signal peptide and brings the signal peptide and its ribosome to the ER Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-21 Ribosome mRNA Signal peptide Signal peptide removed Signalrecognition particle (SRP) CYTOSOL ER LUMEN Translocation complex SRP receptor protein ER membrane Protein Types of Mutations Mutations are changes in the genetic material of a cell or virus Point mutations are changes in just one base pair of a gene The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-22 Wild-type hemoglobin DNA Mutant hemoglobin DNA C T T C A T 3 5 3 G T A 5 G A A 3 5 mRNA 5 5 3 mRNA G A A Normal hemoglobin Glu 3 5 G U A Sickle-cell hemoglobin Val 3 Types of Point Mutations Point mutations within a gene can be divided into two general categories Base-pair substitutions Base-pair insertions or deletions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-23a Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G 5 3 3 5 U instead of C 5 3 Stop Silent (no effect on amino acid sequence) Fig. 17-23b Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end T instead of C 5 3 3 5 A instead of G 3 5 Stop Missense Fig. 17-23c Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of T 3 5 5 3 U instead of A 5 3 Stop Nonsense Fig. 17-23d Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end Extra A 5 3 3 5 Extra U 5 3 Stop Frameshift causing immediate nonsense (1 base-pair insertion) Fig. 17-23e Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 5 3 3 5 missing 5 3 Frameshift causing extensive missense (1 base-pair deletion) Fig. 17-23f Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 5 3 3 5 missing 5 3 Stop No frameshift, but one amino acid missing (3 base-pair deletion) Substitutions A base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code Missense mutations still code for an amino acid, but not necessarily the right amino acid Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Insertions and Deletions Insertions and deletions are additions or losses of nucleotide pairs in a gene Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation These mutations can change the protein much more than a substitution does, and can be disastrous Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Comparing Gene Expression in Bacteria, Archaea, and Eukarya Bacteria have a terminator sequence, archaea and eukarya do not Bacteria (and probably archaea) can transcribe and translate the same gene at the same time Eukaryotes must do these steps separately, because transcription happens in the nucleus and translation happens in the cytoplasm Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-24 RNA polymerase DNA mRNA Polyribosome RNA polymerase Direction of transcription 0.25 µm DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) What Is a Gene? Revisiting the Question The idea of the gene itself is a unifying concept of life We have considered a gene as: A discrete unit of inheritance A region of specific nucleotide sequence in a chromosome A DNA sequence that codes for a specific polypeptide chain Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-25 DNA TRANSCRIPTION 3 RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase NUCLEUS Amino acid CYTOPLASM AMINO ACID ACTIVATION tRNA mRNA Growing polypeptide 3 A Activated amino acid P E Ribosomal subunits 5 TRANSLATION E A Codon Ribosome Anticodon In summary, a gene can be defined as a piece of DNA that can be expressed to produce a functional protein and is inherited by organisms from their parents Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings