PF1684 has RNA-binding activity

To identity which protein has RNA-binding activity, we cut the plasmid using BglII. The self-ligation product, which does not include the region of gene PF1684 and PF1685, has lost the RNA-binding activity (data not shown). This shows the activity is inside these two genes. Then proteins expressed from these two genes were tested for RNA-binding activities and the activity was found only in PF1684 protein (Figure 3 left). The protein was purified to near homogeneity as a 28kDa polypeptide (column 0.5$\mu$ g and 1$\mu$ g in Figure 3 right).

Table 1: Systematic Screening Results from Pyrococcus furiosus Expression library

First Round Candidates				Second Round Candidates
		band	migration	MPOR27	ssRNA15	MPOR27	MPOR27	MPOR27	MPOR27
Number	comments	darkness	length	8/30/04	8/31/04	9/17/04/	9/27/04	10/1/04	11/26/04
#37	? Nuclease			L or x	x
#41		Low	Average	$\circ$ M	$\circ$ H	$\circ$ H	$\circ$ M
#65		Medium	Average	$\circ$ H	$\circ$ H	$\circ$ M	$\times$		$\circ$ H
#73		Medium	Average	$\circ$ L	$\circ$ M	$\times$
#81		Low	Average	$\circ$ L	$\times$
#83		Medium	Average	$\circ$ M	$\circ$ M	$\times$
#98		Low	Average
#134	Thy-1
#194		Medium	Average	$\circ$ M	$\circ$ H	$\times$			$\times$
#211	Fau-1
#307		Medium	Long	$\circ$ H	$\times$	$\times$
#374		Low	Long		$\circ$ M	$\times$
#404		Medium	Long				$\circ$ M	$\times$
#425		High	Average				$\circ$ H	$\circ$ M
#463		High	Long				$\circ$ H		$\times$
#517		High	Long				$\circ$ H	$\times$
#541		Medium	Average				$\circ$ L	$\times$
#591		Medium	Average					$\times$	$\circ$ M
#615		High	Short					$\circ$ M
#618		High	Long					$\times$
#658		High	Long					$\circ$ M

Figure 3: Left:PF1684 RNA-binding activity Confirmation. Right:SDS-PAGE analysis of purified PF1684 protein

Arrow indicates the band of RNA-binding. Free on the bottom is for the free probe (MPOR27). M: marker; 0.5$\mu$ g,1$\mu$ g indicate different amount of the purified PF1684 protein.