Tex 25mer ssrna Binding Stoichiometry

Transcription

1 Figure S. Determination of Tex:2nt ssrna binding stoichiometry using fluorescence polarization. Fluorescein labeled RNA was held at a constant concentration 2-fold above the K d. Tex protein was titrated in at increasing concentration. An inflection point representing the molar ratio where the binding sites have become saturated occurs at equimolar concentrations of Tex and 2nt ssrna indicating a : binding stoichiometry. The assay was performed in mm Tris 7., mm NaCl, % glycerol and measured using a Tecan fluorimeter. 2 Tex 2mer ssrna Binding Stoichiometry 9 mp (arbitrary units) [Tex]/[RNA] (mm)

2 Figure S2. Tex does not appear to have nuclease activity. WT Tex was assayed in a time course experiment in parallel with RNase T, Micrococcal Nuclease, and the Tex YqgF domain active site mutant E33A/D42A. In the assay, the Tex proteins were at a molar concentration x that of Micrococcal Nuclease. Substrate used in this assay is a radio-labeled 3 nt single-stranded RNA and the assay was performed in mm Tris 7., mm NaCl, % glycerol, 3mM MgCl2, mm CaCl2 at 37 C. Reactions were quenched by the addition of an equal volume of phenol/chloroform ph 6.6 and 2mM EDTA. Samples were spun down and the aqueous phase extracted and mixed with 2x formamide loading dye. Samples were run on a 2% acrylamide (9:)/7M Urea denaturing gel in TBE buffer and the gel was scanned using a TYPHOON imaging system (GE Healthsciences). Time (min) No Protein RNase T Micrococcal Nuclease WT Tex E33A/D42A Tex

3 Figure S3. Predicted sites of sequence insertion for Spt6, with respect to the Tex structure. Front (A) and back (B) stereo views of the Tex structure. Highlighted regions (purple) indicate sites where additional sequence exists in Spt6 when aligned with Tex sequences. All highlighted regions lie on the surface of the Tex structure and appear to be able to accommodate additional sequence without disrupting the core structural scaffold. A B

4 Figure S4. Amino acid sequence alignment of Tex (P. aeruginosa) and Spt6 (S. cerevisiae). Observed Tex secondary structure is indicated above the Tex sequence. Highlighted regions indicate identical (red) and similar (yellow) residues. Sequence alignment was performed using ClustalW. Figure was created using ESPript Spt6-Full_Length-S._cerevisiae M D Y K D D D D K T R E E T G D S K L V P R D E E E I V N D N D E T K A P S E E E E G E D V F D S S E E D E D I D E D E D E A R K V Q E G F Spt6-Full_Length-S._cerevisiae 7 I V N D D D E N E D P G T S I S K K R R K H K R R E R E E D D R L S E D D L D L L M E N A G V E R T K A S S S S G K F K R L K R V G D E G N Spt6-Full_Length-S._cerevisiae 4 A A E S E S D N V A A S R Q D S T S K L E D F F S E D E E E E E S G L R N G R N N E Y G R D E E D H E N R N R T A D K G G I L D E L D D F I Spt6-Full_Length-S._cerevisiae 2 E D D E F S D E D D E T R Q R R I Q E K K L L R E Q S I K Q P T Q I T G L S S D K I D E M Y D I F G D G H D Y D W A L E I E N E E L E N G N H M D S I N T R I A E E L S A L P S Spt6-Full_Length-S._cerevisiae 28 D N N E A E E E E I D E E T G A I K S T K K K I S L Q D I Y D L E D L K K N L M T E G D M K I R K T D I P E R Y Q E L R A G I T D Y G N M S consensus> M.... T. I. E.... L H2 H3 H G R V Q P Q Q V A A A V A L L D E G S T V P F I A R Y R K E V T G S L Spt6-Full_Length-S._cerevisiae 3 S E D Q E L E R N W I A E K I S V D K N F D A N Y D L T. E F K E A I G N A I K F I T K E N L E V P F I Y A Y R R N Y I S S R E K D G F L L consensus> A E... V P F I.. Y R. #... S L H H6 H7 3 D D T Q L R M L E E R L R Y L R E L E E R R G A I L A S I E E Q G..... K L T P E L A R D I K L A D T K T R L E D L Y L P Y K Q K R.. Spt6-Full_Length-S._cerevisiae 42 T E D D L W D I V S L D I E F H S L V N K K D Y V Q R F Y A E L H I D D P I V T E Y F K N Q N T A S I A E L N S L Q D I Y D Y L E F K Y A N consensus>7. #. # L L. #....!..... E # L # D. Y..... K H8 H9 H R T K G Q I A L E A G L G A L A D A L F D D P. T L V P E S E A A R F V D A E K G F A D V K..... Spt6-Full_Length-S._cerevisiae 49 E I N E M F I N H T G K T G K K H L K N S S Y E K F K A S P L Y Q A V S D I G I S A E D V G E N I S S Q H Q I H P P V D H P S S K P V E V I consensus> # L #......! K H H2 β A V L E G A K Y I L M E R F A E D A T L L D K L R V F M K N E A T L T A R V V P.... G K E Q E G A Spt6-Full_Length-S._cerevisiae 6 E S I L N A N S G D L Q V F T S N T K L A I D T V Q K Y Y S L E L S K N T K I R E K V R S D F S K Y Y L A D V V L T A K G K K E I Q K G S L consensus> # #.... # K. R # β2 H3 β3. H4 28 K F S D Y F E H D E P L K S A P S H R A L A I F R G R N E G V L S A S L K V G. E E A P G T L H P. C E V M IA E R F G L S N Q G R A A D K Spt6-Full_Length-S._cerevisiae 63 Y E D I K Y A I N R T P M H F R R D P D V F L K M V E A E S L N L L S V K L H M S S. Q A Q Y I E H L F Q I AL E T T N T S D I A I E W N N consensus> %.. # E..... S. K E.... S #..... # H β4 276 W L A E V V R W T W K V K L Y T H L E T D L F G E L R D G A E D E A I S V F A R N L H D L L L A A P A G P R A T L G L Spt6-Full_Length-S._cerevisiae 699 F R K L A F N Q A M D K. I F Q D I S Q E V K D N L T K N C Q K L V A K T V R H K F M T K L D Q A P F I P N V R D P K I P K I L S L T C G Q consensus> %..... #... # L.... # L.. A P.. P T. G

5 β β6 H6 β7 33 D P G L R T G V K V A V V D A T G K L L D T A T V Y P H A P K N Q.... W D Q T L A V L A A L C A K H Q V E L I A I G N G T A Spt6-Full_Length-S._cerevisiae 768 G R F G A D A I I A V Y V N R K G D F I R D Y K I V D N P F... D K T N P E K F E D T L D N I I Q S C Q P N A I G I N G P N P K T Q K F Y consensus> !.... V #.. G ! #..... L Q. #. I. I H7 β8 H8 H9 H2 H2 39 S R E T D K L A G E L I K K Y P G M K L T K I M V S E A G A S V Y S A S E L A A K E F P E L D V S L R G A V S I A R R L Q D P L A E L V K I Spt6-Full_Length-S._cerevisiae 83 K R L Q E V L H K K Q I V D S R G H T I P I I Y V E D E V A I R Y Q N S E R A A Q E F P N K P P L V K Y C I A L A R Y M H S P L L E Y A N L consensus>7. R.. #. L.... I.... G..... I. V. #.. A.. Y.. S E. A A. E F P # !.. A R. $.. P L. E H22 H23 β9 H24 H2 46 E P... K S I G V G Q Y Q H D V S Q L K L A R S L D A V V E D C V N A V G V D V N T A S..... A A L L A R I S G L N S T L A Q N I V A Spt6-Full_Length-S._cerevisiae 9 T S E E V R S L S I H P H Q N L L S S E Q L S W A L E T A F V D I V N L V S V E V N K A T D N N Y Y A S A L K Y I S G F G K R K A I D F L Q consensus> S..!... Q... S... L... L #.... D. V N. V. V # V N. A A.. L.. I S G..... A. # H26 H27 β H28 H29 27 H R D A. N G A F R T R D E L K K V S R L G E K T F E Q A A G F L R V M N G D N P L D A S A V H P E T Y P L V Q R I AA Spt6-Full_Length-S._cerevisiae 97 S L Q R L N E P L L A R Q Q L I T H N I L H K T I F M N S A G F L Y I S W N E K R Q K Y E D L E H D Q L D S T R I H P E D Y H L A T K V AA consensus>7.. #.. N..... R # # L..... L.... F. #. A G F L.! #. L D...! H P E. Y. L...! A A H H3 H32 H33 86 D T E R D I R S L I G D.. S A F L K R L D P K K F T D E T F G L P T V T D I L K E L D K P Spt6-Full_Length-S._cerevisiae 4 D A L E Y D P D T I A E K E E Q G T M S E F I E L L R E D P D R R A K L E S L N L E S Y A E E L E K N T G L R K L N N L N T I V L E L L D G consensus>7 D A. L.. L #... %. # E L..... I.. E L H34 β β β3 H3 63 G R D P R P E F K T A E F Q E G V E S L K D L K P G M V L E G V V T N V T N F G..... A F V D I G V H Q D G L V H I S A L S E K F V K D Spt6-Full_Length-S._cerevisiae F E E L R N D F H P L Q G D E I F Q S L T G E S E K T F F K G S I I P V R V E R F W H N D I I C T T N S E V E C V V N A Q R H A G A Q L R R consensus>7.. #. R. # F... #. # E.. # S L G.!.. V #.. V H36. β4 β 69 P Y. E V V K A G D I V K V K V M E V D I P R N R V G L S M R M S D T P G E K V E G Q R G G R P T G S. G Q P R Q E R G A P R G Q S A P P A Spt6-Full_Length-S._cerevisiae 8 P A N E I Y E I G K T Y P A K V I Y I D Y A N I T A E V S L L D H D V K Q Q Y V P. I S Y S K D P S I W D L K Q E L E D A E E E R K L M M A consensus>7 P.. E!... G..... K V..! D S $... D... #. V #... A A N N A M A A L F A N A K Q L K K K Spt6-Full_Length-S._cerevisiae 24 E A R A K R T H R V I N H P Y Y F P F N G R Q A E D Y L R S K E R G E F V I R Q S S R G D D H L V I T W K L D K D L F Q H I D I Q E L E K E consensus>7 # Spt6-Full_Length-S._cerevisiae 324 N P L A L G K V L I V D N Q K Y N D L D Q I I V E Y L Q N K V R L L N E M T S S E K F K S G T K K D V V K F I E D Y S R V N P N K S V Y Y F Spt6-Full_Length-S._cerevisiae 394 S L N H D N P G W F Y L M F K I N A N S K L Y T W N V K L T N T G Y F L V N Y N Y P S V I Q L C N G F K T L L K S N S S K N R M N N Y R consensus> References. Higgins D., Thompson J., Gibson T., Thompson J.D., Higgins D.G., Gibson T.J. (994). CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. (999). ESPript: multiple sequence alignments in PostScript. Bioinformatics, 3-38.