Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1966 Studies on structure of E coli ribosomes David Lloyd Weller Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons Recommended Citation Weller, David Lloyd, "Studies on structure of E coli ribosomes " (1966). Retrospective Theses and Dissertations. 2923. https://lib.dr.iastate.edu/rtd/2923 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. This dissertation has been microfihned exactly as received 66—10,446 WELLER, David Lloyd, 1938- STUDIES ON STRUCTURE OF E. COLI RIBOSOMES. Iowa State University of Science and Technology Ph.D., 1966 Chemistry, biological University Microfilms, Inc., Ann Arbor, Michigan STUDIES ON STRUCTURE OF E. COLI RIBOSOMES by David Lloyd Weller A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Subject: Biochemistry Approved ; Signature was redacted for privacy. Signature was redacted for privacy. H d 6f Major Department Signature was redacted for privacy. Iowa State University Of Science and Technology Ames, Iowa 1966 il TABLE OF CONTENTS Page ABBREVIATIONS AND SYMBOLS iii INTRODUCTION 1 METHODS AND MATERIALS 12 Preparations of Ribosornes 12 Sucrose Gradient Centrifugation 13 Isolation of RNA 14 Processing of Bentonite 15 Preparation of Bentonite-Agar Gel 16 Physical Methods 17 Chemical Methods 18 Reagents and Special Chemicals 19 RESULTS 21 The Effects of Removing Mg''"^ from Ribosomes 21 Properties of the 30*S Particles 38 Interaction of Bentonite with Various Components 66 DISCUSSION 94 Behavior of Ribosomes at Low Mg"*"^ 94 Interaction of Bentonite with Various Components 104 SUMMARY 110 BIBLIOGRAPHY 114 ACKNOWLEDGMENTS 119 iii ABBREVIATIONS AND SYMBOLS • [-] specific rotation at a particular wavelength, X = «1 observed rotation divided by the product of the concentration of solute (g/100 ml) and the length of light path (decimeters) Â Angstrom = 10~® centimeters N hydrodynamic parameter = ( 1.62^2x10*^ BAG = bentonite-agar-gel DNase = dexoxyribonucle ase pO.1% ^260 = extinction coefficient = absorbency of a solution containing 1 mg RNP/ml, in a cell 1 cm in thick­ ness at 260 my E(P) = molar extinction of free nucleic acids or compon­ ents containing nucleic acid in terms of moles of phosphorus at 260 my = average extinction at 260 my per mole of nucleotide for free RNA EDTA = ethylenediamine tetraacetate sodium salt pH 7.4 f = frictional ratio = ratio of experimentally deter­ fo mined frictional coefficient (f) to the frictional coefficient of a sphere of equivalent volume = 1.0 for an inert sphere and increases the more asymmetrical the particle g = acceleration due to gravity =980 cm/sec^ [n] = intrinsic viscosity = reduced viscosity corrected for concentration M = concentration in moles/liter mM = concentration in millimoles/liter M.W, = molecular weight mg = milligrams ml = milliliter my = millimicron = 10"^ cm iv V = viscosity increment = — = 2.5 for an (1-Vp) inert sphere and increases for assymmetrical particles N = Avagados number = 6.02 x 10^^ molecules/mole p = density of solution = density of water at 20® = 1.00 g/ml PCA = perchloric acid RNA = ribonucleic acid r-RNA = ribosomal RNA RNase = ribonuclease RNP = ribonucleoprotein rpm = revolution per minute S = Svedberg, a unit of sedimentation velocity 10-13 seconds S2 0,w = sedimentation coefficient in Svedbergs reduced to viscosity and density of water at 20° S20 w ~ sedimentation constant = sedimentation coefficient ' corrected for concentration TCA = trichloroacetic acid Tris = tris(hydroxymethyl)aminomethane V = partial specific volume in ml/g = increase in volume resulting from the addition of 1 g of RNP to a large volume of solution 1 INTRODUCTION The mechanism by which amino acids in the cell are poly­ merized into a specific protein involves a small organelle— the ribosome. How the ribosome functions in the biosynthesis of proteins is as yet unknown and the main reason for this is the lack of knowledge concerning the structure of the particle. Studies reported in this dissertation were designed to examine the behavior of the ribosome under artificial conditions in the hope of furthering our knowledge and understanding of the structure of the ribosome. A review of the physical and chemical properties of the ribosomes from the bacterium E. coli will be presented since these ribosomes were employed in the studies reported. Ribosomes are ribonucleoprotein particles which are com­ posed of 60-65 percent RNA and 35-40 percent protein (Tissieres et , 1959). The structure of the ribosomes as revealed by the work of Tissieres et a^. (1959), Hall and Slayter (1959), and Huxley and Zubay (1960) is schematically represented in Figure 1. A 70S ribosome (molecular weight = 2.86 X 10®) is composed of one 30S subunit (molecular weight = 0.85 X 10®) and one 50S subunit (molecular weight = 1.8 x 10®) with the lOOS particle being formed by the dimerization of two 70S ribosomes through the 30S moieties. The stochiometry of the reactions and the molecular weights of various ribosomal particles are shown in the middle and lower rows, respectively, Figure 1. A schematic of the structure of the ribosomes from E. coli 3 a ^ o m cD asos) + 2(50S) •„ 2(70S) KlOOS) M.W. X 10r6 0.85 1.8 2.86 55 4 in Figure 1. Magnesium reversibly effects the association of the ribosomes with all four components being observed at the appropriate concentration^ in 10"^ mM Mg"*"^ only 30S and 50S subunits are observed while increasing the Mg"'"^ concentration to 10 mM results in the formation of 70S and lOOS particles. The 5OS subunit when observed in the electron microscope either combined with the 30S in the 70S ribosome or free in solution is nearly spherical in shape with a diameter of about 160-180 Â. However, the 30S is more asymmetrical and when observed combined with the 5OS appears as a more flattened structure having dimensions of about 70 x 170 Â with the smaller dimension lying along the major axis of the 70S particle. When the 30S is observed free in solution, the sub- unit assumes many different shapes, triangular, trapozoidal, crescent, etc. In some laboratories, cell-free extracts of E. coli pre­ pared in Mg"^^ sufficient to stabilize the lOOS particle contained in addition to the four main components mentioned, an 85S particle and a small amount of a 2OS component (Bolton et al., 1959). The 85S particle is presumed to be a 70S ribosome which is sedimenting faster because of a change in shape and/or hydration while examination of the 2OS material revealed two components, one rich in RNA and the other rich in protein (Roberts, 1960). Kurland (1960) extracted the RNA from the 70S ribosome and the purified subunits and showed the 30S particle 5 contained one molecule of 16S RNA (molecular weight = 0.56 X 10®) while most 50S subunits contained one molecule of 23S RNA (molecular weight = 1.12 x 10®)(Figure 2). However, a few 5OS particles contained two molecules of 16S RNA. Green and Hall (1961) examined the 50S ribosomes and found those 50S subunits that did not form 70S ribosomes in 10 mM magnesium contained 16S RNA while the 50S subunits derived from the dissociation of the 70S particle contained 23S RNA. The structure of free ribosomal RNA has been studied extensively by a number of physical methods and the results of the investigations by Littauer (1961), Cox and Littauer (1962), Kisselev et (1961), Spirin (1960 and 1961) , Hall and Doty (1959), Doty et (1959), Fresco et al. (1960), Spirin and Milman (1960), Boedtker et (1962), and Stanley and Bock (1965) can be summarized as follows: each RNA molecule (16S or 23S) exists as a single continuous polynucleotide chain with a number of helical regions which are formed when the polynucleotide chain loops back on itself. These double stranded regions were originally thought to be stabilized entirely by hydrogen bonds between base pairs, adenine-uracil and guanine-cytosine, but more recent experiments have sug­ gested interactions in addition to hydrogen bonds may be important in maintaining the double stranded regions (Tinco et al.r 1963; Pasman ^ al., 1964). The structure of RNA (secondary and tertiary) under a specific ionic condition and temperature is the result of the interplay among molecular Figure 2. An illustration of the type of RNA extracted from the 30S and 50S ribosomes 7 RIBOSOMES : 30S 50 S M.W. xIO"® 0.85 (Q)"®1.8 •f Ff H RNA-- 16 S 2(163) 23S M.WxlO'® 0.56 1.12 8 forces—the most important of which are the hydrogen bonds between bases and the electrostatic repulsion force between unshielded phosphate groups of the RNA chain. Thus in low ionic strength solutions (water) the double stranded regions are destabilized at room temperature and RNA exists as a long single stranded polynucleotide chain while at a higher ionic strength (about 10"^ M salt) where the salt shields the nega­ tive changes on the phosphate groups, the helical regions are stabilized and the RNA assumes a highly compact coil conforma­ tion. Spirin's laboratory has indicated that in the intermediate ranges of ionic strength the RNA forms a rod- shaped structure in which the double stranded regions are ordered, but other laboratories (Hall and Doty, 1959) contend the RNA exists as a more highly expanded coil which lacks a definite tertiary structure in these solutions. Much less is known about the structure of the RNA in the ribosome.