Journal: Frontiers in Microbiology

Article Title: Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

doi: 10.3389/fmicb.2017.01597

Figure Lengend Snippet: Cold-responsive transcription and translation profiles of the TRAM encoding genes in Methanolobus psychrophilus R15. (A) Quantitative PCR-determined cold-shock induced transcription of four TRAM genes. Three batches of the middle exponential culture grown at 30°C were 4°C-cold shocked for the indicated time before total RNA was extracted. Using the specific PCR primers listed in Supplementary Table and the PCR thermocycler parameters described in section “Material and Methods,” the cDNA fragment was amplified. The transcript copy number of each TRAM was calibrated against its calibration curve and normalized against the 16S rRNA copies. The mean and standard deviation are shown. ∗ Statistically significant difference verified by Student’s t -test ( P < 0.05). (B) Assays of the purified His6-tagged recombinant TRAM proteins on Tricine-SDS-PAGE. (C) Recognition of TRAM2002 and TRAM3066 antibodies to the proteins detected by Western blot. Numbers on the top of gels represent the four TRAMs. (D) Cold-responsive translation profiles of the four TRAMs. Each 100 μg of total protein from strain R15 that was grown at 30 and 18°C, and the 4°C-cold shocked 18°C-culture was electrophoresed using a gradient (10–16%) Tricine-SDS-PAGE. The gel was cut in the lane middle that loaded with a mixture of each 10 ng of His6-tagged TRAM2002 and TRAM3066 (lanes 2002 and 3066), and respectively transferred to 0.1-μm nitrocellulose filter membrane. Using the antibodies against TRAM2002 (left) and TRAM3066 (right), Western blot probed the abundance of the four TRAM proteins. The signals of TRAM2002 and TRAM3066 (Lane 2002 and 3066) could only be seen when extend exposure time (data not shown). Arrows indicted TRAM2002 (1), TRAM3043 (2) and a mixture of TRAM 3066 and TRAM0643 (3) by referencing markers and their molecular weights. CS, cold shocked.

Article Snippet: Total cellular protein was electrophoresed on Tricine-SDS-PAGE below 100 mA for 1.5 h and transferred to a 0.1-μm nitrocellulose filter membrane (Easybio, Beijing, China) under 100 V for 1 h. To fix the proteins to the membrane surface, ddH 2 O and 1 × TBS (2.42 g Tris, 8.0 g NaCl in 1 L, pH 7.6) were used to wash the membrane with gentle shaking for 5 min, and 0.2% glutaraldehyde diluted in 1 × TBS was used to wash the membrane for 45 min.

Techniques: Real-time Polymerase Chain Reaction, Amplification, Standard Deviation, Purification, Recombinant, SDS Page, Western Blot, Membrane