The Shape of Bound Polysomes A. Kent Christensen Department of Cell and Developmental Biology University of Michigan Medical School

Polysome diagram Polysome diagram. From Alberts et al., 1994, 3rd ed, page 108, fig 3-20. 1994I26(7)P.jpg. 72(1076x700)6.

ER 3D diagram Three-dimensional drawing of the endoplasmic reticulum, showing its rough and smooth parts. Bound polysomes are attached to the surface of the rough ER membrane. The artist has drawn many ribosomes bound to the RER membrane, but only a few of them are drawn as being in polysomes (linear sequences of ribosomes arranged in some pattern). Actually, the vast majority of bound ribosomes would be in polysomes. From Alberts et al., 1989, 2nd ed, page 435, fig 8-37. 1990J15(49).jpg. 72(882x700)6.

Polysome surface views How the shape of bound polysomes can be observed occasionally in conventional electron micrographs, when the RER membranes happen to lie obliquely in the section and the polysomes can thus be seen in surface view. From Christensen and Bourne 1999, Anat Rec 255:116-129, fig 1. AKC, rendered by an artist in U-M Biomedical Illustration. 1999C+Bf1.gif. 72(1087x829).

Some bound polysome shapes Some shapes seen in surface views of bound polysomes in electron micrographs. ShapeDiagr.gif. 72(463x800).

These shapes were first described by George Palade in his original description of ribosomes George E. Palade, 1955. A small particulate component of the cytoplasm. J Biophys Biochem Cytol 1:59-68. Particles mostly 100-150A in diameter. Particular affinity for membranes of ER, but also free. Surface views of RER described on page 61: "... 'attached' granules are frequently disposed in linear series and that in these series they are spaced at more or less regular intervals, i.e., 80 to 150 A. The linear series in turn form consistent patterns, among which parallel double rows, loops, spirals, circles, and rosettes (Figs 3 and 5) appear to be predominant. Although such patterns are of frequent occurrence, and, moreover, seem to recur preferentially in particular combinations for a given cell type..."

SEM of bound polysomes (Tanaka) SEM of bound polysomes in a rat motor neuron. Some of the polysomes seen here have a spiral shape, although there are also partial circles. SEM kindly provided by Dr. Keiichi Tanaka, M.D., Department of Anatomy, Tottori University School of Medicine, Yonago 683, Japan. It was taken with an ultrahigh resolution SEM capable of resolving about 5 A. The tissue had been fixed by perfusion with glutaraldehyde/formaldehyde, postfixed with osmium tetroxide, treated with DMSO, freeze-cleaved, then given further osmium treatment. A polysome similar to those show here, but at much higher magnification, can be seen in Tanaka et. al., 1989, J Electron Microsc Technique 12:146-154, fig 4.

Model of a polysome based on a cryo-EM reconstruction of the prokaryotic ribosome (Frank) Model of a prokaryotic polysome, showing the detailed structure of the ribosomes and their relationship to the mRNA. Blue = large ribosomal subunit, yellow = small ribosomal subunit, red = mRNA, green = part of a tRNA, brown = nascent polypeptide. Although this prokaryotic polysome has been drawn as a circle, the living polysome would probably not be restricted to that shape, and so would not be a circular polysome in the sense used in this presentation. Polysomes in a prokaryote, such as a bacterium, are free in the cytoplasm, and can thus assume a variety of shapes in 3-dimensions. This would also be true for the abundant free polysomes that occur in eukaryotic cells. On the other hand, eukaryotic polysomes that are tightly bound to the membranes of the rough endoplasmic reticulum are confined to the 2-dimensions of that membrane surface. These bound polysomes can be maintained in a particular shape, such as circles, spirals or hairpins -- as is shown in the studies described in this presentation. This striking figure was kindly provided by Dr. Joachim Frank, Howard Hughes Medical Institute, Health Research, Inc., at the Wadsworth Center, Empire State Plaza, Albany, New York 12201. It is copyrighted but as yet unpublished, and is used here with Dr. Frank's permission. The way the ribosomes are strung on the mRNA was based on the appearance of some polysome preparations in electron micrographs. The detailed reconstruction of the ribosomes was based on cryo-electron microscopy of unfixed, unstained and fully hydrated ribosomes in a thin layer of ice, followed by computerized 3-dimensional reconstruction from large numbers of images. For further details and references see Frank, 2001, BioEssays 23:725-732.

Do bound polysomes for a particular protein have a consistent shape? Study bound polysome surface views in a cell type that makes large quantities of a particular secretory or membrane protein. The great majority of observed bound polysomes will be for that protein. Cell type Product M.W. (kDa) Shape Ref Somatotrope Growth hormone 22 Circle, 6-7 rib 1987 Mammotrope Prolactin 23 Thyroid epithelium Thyroglobulin subunit 330 Hairpin, ~92 rib 1999 Fibroblast Collagen I a-chain 150 Hairpin, ~48 rib Retinal rod Opsin (rhodopsin) 354 a.a. 7 t.m. Spiral, ~9 rib Abstract 1998

Growth hormone and prolactin Christensen AK, Kahn LE, Bourne CM, 1987. Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. Am J Anat 178:1-10

Pituitary, EM EM of rat pituitary, showing somatotrope (source of growth hormone), mammotrope (prolactin), and a gonadotrope (luteinizing hormone and follicle stimulating hormone). Taken by Larry E. Kahn, in AKC lab, about 1980. LEK11-06-17L.jpg. 72(718x700)6.

Somatotrope cytoplasm, EM Polysomes in somatotrope cytoplasm. This cell type secretes growth hormone in large quantities. The rough ER is seen edge-on at right, and becomes more in surface view as you move to the left. The preponderant polysome is a small circle. Christensen et al 1987, fig 2. C+1987(2). CB 1.20.21.81. 1987C+f2.jpg. 72(949x700)6.

Circular polysomes, grid Circular polysomes from Mammotropes (PRL) and Somatotropes (GH). From Christensen et al 1987, fig 4. 87grid.jpg. 1987grid-2.jpg. 72(778x700)6.

Circular polysome, detail Detail of circular polysome for growth hormone and for prolactin. From Christensen et al 1987, fig 4 (slightly modified). The ribosomes are shown in the diagram approximately as they appear in electron micrographs (at right). Arrowheads indicate strands often seen connecting one ribosome with the next. The strand may be the mRNA (perhaps enlarged by internal base pairing and protein binding). The small subunit is shown on the inside of the mRNA curve. Further evidence for the identity of the strand and the orientation of the small subunit will be given later in the talk. CircleDiagr.jpg. 72(425x400)6. 1987detail.jpg. 72(658x400)6

Spiral "G" polysome The spiral "'G" polysome is another characteristic polysomal shape seen occasionally in somatotropes (above white line) and in mammotropes (below white line). We have no idea what protein it would make. From Christensen et al 1987, fig 4. 1987C+f4b.jpg. 72(157x700)6.

Size distribution of circular and spiral G polysomes Graphs showing size distribution of circular and spiral G polysomes in somatotropes (growth hormone) and mammotropes (prolactin). From Christensen et al 1987, fig 5. 1987C+f5.gif. 72(1100x622)

Thyroglobulin subunit and a-chains of collagen I Christensen AK, Bourne CM, 1999. Shape of large bound polysomes in cultured fibroblasts and thyroid epithelial cells. Anat Rec 255:116-129.

Cultured fibroblast, EM EM of cultured fibroblast, sectioned in the plane of the cell (to maximize the occurrence of polysome surface views). From Christensen and Bourne 1999, fig 2. 1999C+Bf2.jpg. 150 dpi (1400 width).

Fibroblast cytoplasm, EM EM of polysome surface views in cytoplasm of a cultured fibroblast. From Christensen and Bourne 1999, fig 4. 1999C+Bf4.jpg. 72(528x700)6.

Detail-1 Upper half of previous figure (fig 4) in more detail. From Christensen and Bourne 1999, fig 4. 1999C+B4a.jpg. 72(995x700)6.

Detail-2 Lower half of figure 4 in more detail. From Christensen and Bourne 1999, fig 4. 1999C+Bf4b.jpg. 72(951x700)6.

Cultured thyroid cell, EM EM of cultured thyroid follicular cells, sectioned in the plane of the cell (to maximize the occurrence of polysome surface views). From Christensen and Bourne 1999, fig 3. 1999C+Bf3.jpg. 72(1104x700)6.

Thyroid cell cytoplasm, EM EM of polysome surface views in cytoplasm of a cultured thyroid follicular epithelial cell. From Christensen and Bourne 1999, fig 5. 1999C+Bf5.jpg. 72(554x700)6.

Detail-1 Upper half of previous figure (fig 5) in more detail. From Christensen and Bourne 1999, fig 5. 1999C+Bf5a.jpg. 72(1034x700)6.

Detail-2 Lower half of figure 5 in more detail. From Christensen and Bourne 1999, fig 5. 1999C+Bf5b.jpg. 72(940x700)6.

Hairpin polysomes, fibroblast Surface views of hairpin polysomes from cultured fibroblasts. The insert is a drawing of the probable appearance of a polysome for an alpha-chain of collagen type I, drawn in the same scale as the electron micrographs. From Christensen and Bourne 1999, fig 6. 1999C+Bf6.jpg. 72(657x700)6.

Detail-1 Upper half of the previous figure (fig 6) in more detail. Arrowheads indicate curved ends of hairpin polysomes. Brackets enclose portions of hairpins where ribosomes in the two strands appear to be in register. The hairpin polysomes seen here are not complete, because their large size causes them to go out of the plane of section. From Christensen and Bourne 1999, fig 6. 1999C+Bf6a.jpg. 72(1048x700)6.

Detail-2 Lower half of figure 6 in more detail. From Christensen and Bourne, fig 6. 1999C+Bf6b.jpg. 72(1064x700)6.

Hairpin polysomes, thyroid Surface views of hairpin polysomes from cultured fibroblasts (7A) and from in vivo thyroid follicles (7B). Christensen and Bourne 1999, fig 7. 1999C+Bf7.jpg. 72(600x700)6..

Detail-1 Upper half of the previous figure (fig 7) in more detail. From Christensen and Bourne 1999, fig 7. 1999C+Bf7a.jpg. 72(1100x592)6.

Detail-2 Lower half of figure 7 in more detail. From Christensen and Bourne 1999, fig 7. 1999C+Bf7b.jpg. 72(985x700)6.

Thyroglobulin polysome Diagram showing the probable appearance of a complete hairpin polysome for a thyroglobulin subunit (330 kDa). From Christensen and Bourne 1999, fig 10. 1999C+Bf10.gif. 72(1400x426).

Spiral & circular, fibroblast Surface views of spiral and circular bound polysomes seen in cultured fibroblasts. The upper two rows show a large spiral that was seen occasionally. The inset diagram indicates the probable appearance of this spiral (in the same scale as the electron micrographs). Some of the spirals and circles have dark particles (indicated by very small arrows) in their center that are larger than ribosomes. It is suggested in the paper that these could be sites of ribosomal pausing. Christensen and Bourne 1999, fig 8. 1999C+Bf8.jpg. 72(922x700)6.

Spiral & circular, thyroid Surface views of spiral and circular bound polysomes seen in cultured thyroid follicular epithelial cells. The top row shows a large spiral polysome seen occasionally, resembling that seen in fibroblasts in the previous figure. From Christensen and Bourne 1999, fig 9. 1999C+Bf9.jpg. 72(1100x507).

Hairpin and spiral compared Comparison between hairpin and spiral polysomes. From Christensen and Bourne 1999, fig 11. 1999C+Bf11.gif. 72(1400x930).

If bound polysomes were randomly arranged Bound polysomes in a hypothetical "random" arrangement. Made by AKC in Photoshop. RandomPolysomes.jpg. 72(1050x700).

How is polysome shape maintained? From Christensen and Bourne 1999. Natural tendency of mRNA to bend. Curved path through ribosome, internal base pairing, interactions with proteins. Postulated spacer component, to maintain distance between strands in larger bound polysomes. Spacer is probably one or more membrane proteins. Ends of proteins may bind to translocons (sec61) under ribosomes. Some kind of spacer may also be necessary to keep polysomes from touching one another.

The mRNA in bound polysomes has a natural tendency to bend From Christensen and Bourne 1999. Isolated small free polysomes, brought down on an EM grid membrane, spontaneously form a circle, with ribosomal small subunits inside the curve. Shelton E, Kuff EL, 1966. J Mol Biol 22:23-31. Same curve and subunit orientation is true for polysomes bound to the rough ER. Christensen AK, 1994. Cell Tissue Res 276:439-444. Curved path of mRNA within each ribosome, with small subunit inside the curve. Agrawal RK et al., 1996. Science 271:1000-1002.

Shelton & Kuff 1966 Small free polysomes brought down on an EM grid membrane and then negatively-stained. Note that the polysomes assume a curved pattern, and that the small subunits are on the inside of the curve. From Emma Shelton & E. L. Kuff 1966, Substructure and configuration of ribosomes isolated from mammalian cells, J Mol Biol 22:23-31. This is from plate II, fig a. Shelton.jpg. 72(938x700). Required considerable Gaussian blur (otherwise distracting moire patterns).

Christensen 1994, models Predicted appearance for negative stain with the traditional model of ribosomal subunit orientation in a bound polysome versus Nigel Unwin's model, which was based on EM 3D reconstruction of ribosome crystals bound to membrane but not involved in protein synthesis (Unwin and Taddei 1977, J Mol Biol 114:491-506; Unwin 1977, Nature 269:118-122). From Christensen 1994, fig 2. 1994Cf2.jpg. 72(1159x700)6. Required considerable Gaussian blur to avoid moire patterns in dark areas. It was also necessary to increase contrast considerably (Image>Adjust>Brightness-Contrast).

Christensen 1994, neg. stain When bound polysomes on microsome vesicles are negatively-stained and viewed in EM (figs a and b), the subunits are visible, supporting the Unwin side-by-side model. The small subunit is on the inside of the curve. In positively-stained tissue polysomes seen in surface view (figures c and d), the strands seen occasionally between ribosomes occupy approximately the same position as the gap between subunits in the negatively-stained polysomes -- giving further evidence that the strands are the mRNA (perhaps thickened by bound protein and internal base pairing). From Christensen 1994, fig 1. 1994Cf1.jpg. 72(548x700)6.

Agrawal et al 1996 Ribosome drawing showing tRNA in A, P, E sites. Note curved path of mRNA (5' and 3'), with small subunit (30S) lying in the curve. 50S subunit (CP=central protuberance, L1=L1 protein), 30S subunit (h=head, ch=channel, sp=spur), white and purple = anticodon binding. From Agrawal et al 1996, Science 271:1000. Agrawalf3.jpg. 72(487x700)6. Gaussian blur necessary to minimize moire patterns.

Postulated spacer component From Christensen and Bourne 1999. A consistent feature seen in larger polysomes, such as hairpins and large spirals, is a relatively constant distance between strands. This suggests some kind of spacer component to maintain this distance. Such a spacer might consist of one or more membrane proteins. If there is such a spacer, then its ends might have binding sites on the ribosomes. However, as can be seen in the next diagram, the ribosomes in adjacent strands of a hairpin polysome face each other ("front-to-front"), while those of adjacent strands in large spiral polysomes are "front-to-back. This would complicate any postulated binding of a spacer to ribosomes. A more likely binding site for the postulated spacer might be on the Sec61 channel that underlies each ribosome, binding the ribosome to the membrane and providing the channel by which nascent secretory proteins pass through the RER membrane. The channel is formed by three Sec61 trimers arranged around a central channel, and if each of those units carried a binding site for the spacer, then the spacer could probably bind from any direction.

Comparison of hairpin & spiral Comparison between hairpin and spiral polysomes. From Christensen and Bourne 1999, fig 11. 1999C+Bf11.gif. 72(1400x930).

Corsi & Scheckman 1996 Mammalian co-translational translocation, showing how the ribosome becomes bound to the Sec61p translocon (red). Corsi and Scheckman 1996, J Biol Chem 271:30299, fig 1. Corsif1.jpg. 72(929x700)6.

Ribosome-Sec61 complex (Beckman et al 1997) Ribosome-sec61p (channel) complex. The channel is in the RER membrane, and consists primarily of Sec61 trimers arranged around a central pore, through which nascent secretory proteins pass through the membrane to reach the RER lumen. From Beckman et al 1997, Science 278:2123, fig 3. CBSbeckmanF3.jpg. 72(691x700)6.

Possibility that ribosomes are linked between strands From Christensen and Bourne 1999. As was mentioned earlier, in hairpin polysomes the ribosomes in the two strands often appear to be in register, suggesting a linkage between ribosomes at the same level in the two strands. Examples are enclosed in thin brackets in figures 6 and 7 of Christensen and Bourne 1999. If there is significant linkage, then it could impose constraints on movement during protein translation. Since translation proceeds in opposite directions in the two strands, then the ribosomes would of necessity be immobile, and only the mRNA could be moving during translation. The 5'-end of the mRNA would thus move successively through all positions in the hairpin. It is possible that the same spacer that maintains distance between strands might also bring about the linking.

Extent of polysomal mRNA contraction From Christensen and Bourne 1999. Average center-to-center distance between ribosomes in a hairpin polysome = ~24.9 nm. Number of nucleotides in that distance = ~90 (Staehelin et al. 1964, Nature 201:264). Thus ~0.28 nm/nucleotide. Length of fully-extended RNA nucleotide = 0.59 nm (Sundaralingam 1974). Therefore polysomal mRNA is about half its fully-extended length in a bound polysome.

Other examples of consistent shape for bound polysomes IgG antibody: circular bound polysomes of 17-18 ribosomes (for heavy chain) and 7 ribosomes (for light chain). Kitani, et al., 1982. Ultrastructural analysis of membrane-bound polysomes in human myeloma cells. Blut 44:51-63. Ovalbumin: spiral of about 13 ribosomes. Palmiter RD, Christensen AK, Schimke RT, 1970. Organization of polysomes from pre-existing ribosomes in chick oviduct by a secondary administration of either estradiol or progesterone. J Biol Chem 245:833-845. Opsin (rhodopsin, integral membrane protein, with 7 transmembrane domains): loose spiral of about 9 ribosomes. Published in abstract: Christensen AK, 1998. The shape of opsin polysomes observed by electron microscopy on the surface of the endoplasmic reticulum in Xenopus retinal rod cells. Mol Biol Cell, vol 9 supplement, page 221a. Poster for annual meeting of the American Society for Cell Biology, San Francisco, 13-17 December 1998. (Abstract).

Retinal rod cell, EM (Christensen 1998 abstract) EM of Xenopus retinal rod cell. Opsin is synthesized on the endoplasmic reticulum of the myoid segment. The arrow indicates the cilium from which the outer segment arises (a similar cilium can also be seen in the neighboring cone cell). From Christensen 1998 abstract. 1998J09(579).jpg. 72(969x700)6.

Myoid ER, rough or smooth? (Christensen 1998 abstract) Ribosomes are so sparse on the myoid ER that it has occasionally been described as being smooth ER. Yet it is the site of opsin synthesis, and so is biochemically a rough ER. The ER may appear to be smooth because most of the ER membrane is occupied by opsin (an integral membrane protein), which is the product of protein synthesis on this ER. So polysomes are situated here and there on the ER membrane, where they compete for space among the opsin molecules. From Christensen 1998 abstract. 1998G22(460).jpg. 92(829x700)6.

Surface views of opsin polysomes (Christensen 1998 abstract) Surface views of opsin polysomes (arrowheads) on the RER in the myoid of a Xenopus retinal rod cell. Gly = glycogen particles, which are common in the cytoplasm but don't attach to the ER. From Christensen 1998 abstract. 1998G22(459).jpg. 72(571x700)6.

Opsin polysomes in surface view (Christensen 1998 abstract) Surface views of opsin polysomes, a loose spiral of about 9 ribosomes. From Christensen 1998 abstract. OpsinMatrixP.jpg. 72(1100x289)6.

Diagram of opsin polysome (Christensen 1998 abstract) Opsin polysome diagram. From Christensen 1998 abstract. OpsinDiagr.jpg. 72(661x700). Required some Gaussian blur (to eliminate moire patterns in ribosomes).

Conclusions Polysomes bound to membranes of the rough ER assume non-random shapes. Bound polysomes making a particular secretory or membrane protein have a consistent shape. The shape is caused by a natural tendency of polysomal mRNA to bend and, for larger polysomes, also probably involves spacer proteins that maintain the distance between mRNA strands.

Co-workers Larry E. Kahn Carol M. Bourne Ursula Reuter Tami B. Grossfield Kjung-Mi Lim Terry B. Lowry Jonathan M. Barkey