The Shape of Bound Polysomes

A. Kent ChristensenDepartment of Cell and Developmental Biology

University of Michigan Medical School

Polysome diagram

ER 3D diagram

Polysome surface views

Some bound polysome shapes

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)

Model of a polysome based on a cryo-EM reconstruction of the prokaryotic ribosome (Frank)

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 Circle, 6-7 rib 1987

Thyroid

epithelium

Thyroglobulin subunit

330 Hairpin, ~92 rib 1999

Fibroblast Collagen I

-chain

150 Hairpin, ~48 rib 1999

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

Somatotrope cytoplasm, EM

Circular polysomes,

grid

Circular polysome, detail

Spiral "G" polysome

Size distribution of circular and spiral G polysomes

Thyroglobulin subunit and -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

Fibroblast cytoplasm,

EM

Detail-1

Detail-2

Cultured thyroid cell, EM

Thyroid cell cytoplasm, EM

Detail-1

Detail-2

Hairpin polysomes, fibroblast

Detail-1

Detail-2

Hairpin polysomes,

thyroid

Detail-1

Detail-2

Thyroglobulin polysome

Spiral & circular, fibroblast

Spiral & circular, thyroid

Hairpin and spiral compared

If bound polysomes were randomly arranged

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

Christensen 1994, models

Christensen 1994, neg.

stain

Agrawal et al 1996

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

Corsi & Scheckman 1996

Ribosome-Sec61 complex (Beckman et al 1997)

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 distancebetween 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)

Myoid ER, rough or smooth? (Christensen 1998 abstract)

Surface views of opsin

polysomes (Christensen

1998 abstract)

Opsin polysomes in surface view (Christensen 1998 abstract)

Diagram of opsin polysome (Christensen 1998 abstract)

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