Traditionally: polymerase tracks along the DNA strand. No scientific evidence, except for:

• logic... small object should move

  analysis difficult

• most complexes inactive (2-6/40)

exp # 1

polymerase with labeled precursors forms nascent DNA attached to substructure. (membrane or nuclear matrix)

Problems: unphysiological .... actually, DNA should had aggregated

DNA synthesis in vitro.

Exp # 2

DNA synthesis in vitro:

12 units helicase expected to split, and track in opposite directions: moving polymerase theory

Actually, helicase remained together.... being each 6 units an attachment to the other.... same geometry must follow for polymerase

exp # 3

replication of mammalian chromosomes. Labeled, new DNA must had been removable with electroeluted chromatin.... it didn't, showing it was attached, probably due to a fixed polymerase

The new DNA was held by the attached polymerase

Replication factories

exp # 4

rat fibroblasts in S phase, labeled new DNA. Sites of incorporation in ~ 150 foci, not diffused, as expected. As chromatin duplicates, there are less foci, but larger. Each foci contains everything necessary for replication.

It has been shown that new DNA is originally attached to electron dense bodies... probably the fixed polymerase

conclusion....

many replication forks need to be working at the same time. Each focus is a factory, with enzymes working on different templates.

Exp # 5

catalytic unit of polymerase labeled. If forks moved independently.... the should has been seen. Only one spot seen. The 4 active polymerase should had been on one site...... proves factory theory

working with RNA

probably the same as with DNA polymerase. Current experiments proving otherwise might allow the polymerase to attach to something. (they are performed with high protein concentrations for a long time). Evidence is they pellet in centrifuge.

Exp # 6 ... miller and the christmas tree

nuclei dropped on hypotonic solution, then photographed shows the polymerase on act, showing it tracks.... however this is a static picture. Also, if done in hypertonic solution, then the new RNA is seen attached to something.... the polymerase, attached to the matrix.

Exp #7

HeLa cells.... labeled RNA doesn't detach when it should (tracking polymerase model).

Other problems

• torsion generated by unwinding: solved by topoisomerase

• entangled transcript, once every 10 base pairs. Untangling must be perfect, otherwise RNA couldn't get to the cytosol.

Problem is solved if polymerase fixed and DNA rotates.

Exp # 8

labeled new RNA. RNA is shown in foci. Adding two different drugs proves sites for polymerase I is different from II and III. II and III also different, others experiments. Both with around the same size 40-80 nm.

Exp # 9 ... factories again

cannot answer in nucleoplasmic transcription (II and III)... too small

the nucleolus produces 45S RNA and ribosomes. It contains 3 zones:

• fibrillar center associated with

• dense fibrillar component

• granular component (where ribosomes mature)

In HeLa cells, ~ 540 45S rRNA genes, tandem repeat, different chromosomes. ~120 genes come together, and form ~ 30 fibrillar centers, each with polymerase I, and 4 active genes close to it (4*30 = 120). Each gene is associated with ~ 125 polymerase. The nucleolar factory contains ~ 500 active polymerase and 4 transcription units.

It would be difficult for the polymerase + transcript to track around the dense fibrillar component... but it;s easier for the DNA to go around it. The DNA must be the branches seen on the chrsitmas tree.

The same is believed to happen with nucleoplasmic transcription. Around 100 000 polymerase must be active in a mammalian cell. In HeLa..... 15,000 P1; 65,000 P2; 10,000 P3.

Most units only associated with one polymerase. It is believed that more than 1 P2 can be working on the same unit, but only sometimes.

Estimates indicate there are more transcripts than sites, and each transcription unit is only associated with 1 polymerase, then each site must contain more than one transcription unit.

Therefore, active RNA polymerases, like the DNA ones, are in factories. The RNA factories would also contain capping, polyadenylatinga and splicing enzymes.

Pulling

exp # 10

binding polymerase of phage to plastic. It produced RNA.

Exp # 11

E. coli polymerase attached to glass slide. Template with gold end. Particles became more still as transcription begun. The transcription rate was around the same as with the "unbound" enzyme.

Conclusion of 10 and 11: the polymerase has enough power to reel in the template and extrude the transcript.

Exp #12

same as 11, polystyrene instead of gold. Enzyme pulled with 14 pN

that's 1/13 force needed to brake H bonds in duplex

larger than force kinesin-myosin.

It moves 0.34 nm, that;s 1/10 step of kinesin-myosin.

Conclusion

evidence for DNA polymerase fixed:

1. theoretical : attached DNA polymerase can be controlled coordinately.

2. Some stuff may become immobilized in vitro.

3. Active DNA polymerase and the nascent strand resist detachment.

4. Newly made DNA is in foci, which means polymerases are not free to track. This also supports the factory idea.

Now the factory idea is more accepted. In eukas, this factories aren't permanent.

The fixed RNA polymerase idea is less accepted. Reasons to believe:

1. logic .... or how to untangle

2. all eukas polymerases assemble into complexes, and pellet in centrifuge

3. the polymerase, transcripts, and active transcription units associated with substructure.

4. New RNA in discrete foci.

Doubt if concentrated in factores

• sure for P1 in eukas.

• More circunstancial evidence supports P2 and P3

if these 2 polymerases are fixed, others should be too. Immobilization can be done by fixing with partners in dimeric complex. (exp #2) , or by attaching to a viral capsoid, cell membrane or internal skeleton. That;s for replication, transcription, and reverse transcription.

All these involve template movement.

Model for this involves initial DNA binding, passage through the fixed polymerization site, detachment. The accesibility of the promoter to the enzyme will determine rate initiation, and this will be affected by proximity to a factory.

Raised questions

• polymerase order inside factories

• firing factors inside a factory, enabling some to fire at the same time

• do transcription factories specialize in one or some genes

• other functions on the factory

• in bacteria, does the polymerase organize the traducing ribosomes

figures

fig 1: shows models for replication/transcription. A) tracking enzyme. Tetramer with 4 polymerases split, and two halves move apart. B) the polymerase makes the transcript. C) fixed polymerase. Daughter strands extruded in loops. D) RNA extruding. Fixed enzyme.

Fig 2 : explains fig1 C. Sites on the old DNA bind to fixed polymerase. In turn, the polymerase extrutes double helix... in loops, semiconservative replication system.

Fig 3: movements of RNA and polymerase to avoid entangling.

A) moving and rotating polymerase: entangling. Could order by pulling, but RNA would break. Rotation of the transcript... number of loops must be exact. Could use cutting-pasting enzymes. Another solution is to attach the transcript to the enzyme... but then the polymerase would have to carry other enzymes... bacteria

B) if polymerase moves, but doesn't rotate. Supercoils formed and easily removed. Difficult to imagine preventing the polymerase to rotate.

C) DNA moving, polymerase rotating. .. same as in A

D) enzyme attached, DNA moves. Supercoils must be removed as in B... only easier to imagine.

Fig 4: just shows models for transcription factory. The nucleoplamsic one more complicated than the nucleolar one

fig 5: transcription cycle, as described in conclusion.