stability of the poly U-AR complex. Poly U ir 

 0.4 M NaCl gave a small positive rotation at 

 low temperature (at 1.5 x 10-^ M, the observed 

 rotation was about 0.2 degree at 5°C). The 

 rotation decreased with increasing tempera- 

 ture, finally became temperature insensitive 

 beyond 12° C as shown in the control curve in 

 Fig. 5. On the otherhand, l.SxlO-^M adenosine 

 alone gave an observed rotation of -0.09° cal- 

 culated from the rotation at 1.2 x lO-^M which 

 was temperature independent. Nevertheless, 

 where the two were mixed, a large increase 

 in positive rotation was observed, +1.03° at 

 5°C. At the temperature insensitive region, 

 the rotation of the mixture was the algebraic 

 sum of its constituents. We took these to mean 

 that the poly U-AR complex formed an ordered 

 structure in 0.4 M NaCl and its stability was 

 reflected by its melting behavior in response 

 to the temperature variation. In 0.4 M salt, the 

 optical rotation measurements remained essen- 

 tially invarient with a temperature range from 

 0.5° C to 20° C. When poly U is mixed with 

 cytidine, inosine or methylated adenosines no 

 complex formation was observed (Fig. 5). 



Formations of poly U-AR complex and its 

 thermostability were highly dependent on adeno- 

 sine concentration as illustrated in Fig. 6, When 

 a constant amount of poly U (1.5 x lO-^M) was 

 allowed to interact with varying amounts of 

 adenosine ranging from 3 x 10"^M to2x lO'^M, 

 a saturation phenomenon similar to that ob- 

 served in the equilibrium dialysis was also 

 found, i.e., the magnitude of the maximum 

 rotation and apparent stability remained un- 

 changed after the ratio of input adenosine per 

 UMP of poly U (denoted by A/U) reached unity. 



Various analogs of adenosine were also 

 tested for their binding capacity to poly U by 

 the optical rotation and sedimentation methods 

 with the expectation of obtaining information 

 about an involvement of binding sites, and the 

 role of the sugar moiety. The following com- 

 pounds were tested: deoxyadenosine, 

 L-adenosine (the pentose was L-ribose instead 

 of D-ribose), (9 r-hydroxypropyl) adenine and 

 (9-hydroxypentyl) adenine, (long-chain alcohols 

 in replacing the sugar moiety). Complexing 

 with poly U was found for all these four com- 

 pounds. When the point of attachment of the 

 purine ring was changed from the 9 position 

 to the 3 position as in the case of 3-isoadenosine, 

 complex formation could still take place. All 

 these observations indicate that the sugar moiety 

 of the adenosine does not play an important 

 role for the binding. Optical rotation studies of 



the mixture of poly U with N-6-methyladenosine, 

 with 1-methyladenosine and with tubercidin 

 ',A pyrrolo 2, 3-d pyrimidine riboside) revealed 

 that no interaction took place. Therefore, the 

 N-6-amino group of the adenineand with tuber- 

 cidin (A pyrrolo 2, 3-d pyrimidine riboside) 

 revealed that no interaction took place. There- 

 fore, the N-6-amino group of the adenine ap- 

 pears to be definitely involved in binding with 

 poly U. Other possible bonding sites are the 

 N-1 and N-7 position of the adenine. 



The two important aspects of the parti- 

 cipation of adenosine in the interaction are its 

 concentration dependence and specificity. The 

 complex formation is undetectable in low nu- 

 cleoside concentration. After a threshold con- 

 centration of adenosine is reached, the binding 



-02 



20 



30 



Fig. 5. 



Observed rotation of poly U (1.5 x iO M ) - nucleoside 

 mixtures versus temperature in 0.4 1/ NaCl, HMP. Con- 

 centrations of the nucleosides are: adenosine (AR), 

 1.5 X 10-^1/ C — • — ) and 7.3 x W^ M < — • --); L- 

 adenosine (L-AR), 9.3 x 10"^ W ; deoxyadenosine (dAR) 

 7.8 X 10-3 ]i ; cytidine (CR), 1.1 x lO-^U; N-6-methyl- 

 adenosine (M^-AR), 7 x 10-3 1/ ; inosine (HxR), 1.5 x 

 10-2 i; . (From Huang and Ts'o, /. Mol. Biol. 16.523, 1966; 

 reproduced with permission of Academic Press.) 



192 



