Having uS13 and uL5 tagged with Venus, we intended to assess whether the presence of tag would alter the growth of SLVNC with respect to Wt. We observed no apparent cell growth defect for SLVNC under the given experimental conditions suggesting that the tag doesn’t impair the growth (Fig. 3.2A). Following this, SLVNC cells were screened for the expression of Venus tagged uS13 and uL5 by RT-PCR (Fig. 3.2B). After confirming the expression by RT-PCR, we also tested for the integration of the Venus
tagged uS13 and uL5 onto ribosomes from SLVNC cells by immunoblotting (Fig. 3.2C). It turns out that about 25% of the ribosomes harbour Venus tagged uS13 and uL5. Upon confirming the expression and integration, we proceeded to test whether the fluorescence complementation would occur in strains other than SLVNC. We quantified the relative fluorescence (rf) that is the ratio of fluorescence from cell lysate of Venus tagged variant to fluorescence from cell lysate of SLVNC (Fig. 3.2E). It turned out that though lysates from SVN and LVC emitted low background compared to SLVNC (rf < 1), other variants such as VNC, SVNC, and LVNC were fluorescing as good as SLVNC (rf ≥ 1). This suggests that even in the absence of uS13 or uL5, the association between VN and VC fragments occurs in a location other than the ribosome leading to fluorescence complementation.
This, in turn, would lead to high background signal if one were to assess the complementation directly from the cell lysate.
Since we were interested in monitoring the association of the Venus tagged ribosomal subunits alone, we reasoned that the background noise could be mitigated if the free Venus tagged uS13 and uL5 were segregated from the ribosome-bound counterparts.
It is worth mentioning that in order to achieve this, it was just sufficient to purify crude ribosomes by clarifying the cell lysate on an isocratic sucrose cushion. Subsequent steps of density gradient centrifugation and fractionation of ribosomal subunits, as performed for ribosomal profile generation, were obviated (vide. Methods). After purifying the crude ribosomes from SLVNC, we tested for the presence of fluorescence signal. Purified crude ribosomes from Wt and VNC were analysed along with SLVNC for the fluorescence signal (Fig. 3.2D). Wt lacks the Venus tag; no complementation is expected to take place on the ribosome for VNC. Therefore, BiFC is not expected from these variants. Indeed, though fluorescence from SLVNClysates was comparable to that measured from VNC lysates (vide.
Fig. 3.2D inset), in the modified approach, no significant fluorescence was observed for crude ribosomes purified from Wt and VNC (Fig. 3.2D). However, crude ribosomes isolated from SLVNC alone exhibited significant fluorescent signal (Fig. 3.2D). This suggests that the background noise can be largely mitigated for BiFC analysis even using crude ribosomes that can be rapidly purified from cell lysates.
Figure 3.2 Ribosomes isolated from BiFC competent strain are fluorescent (A) A growth comparison between the Wt and SLVNC strains is shown.
(B) Confirmation of expression of the cloned constructs was done using RT-PCR.
The marker is shown in the middle of the gel and the corresponding sizes of the bands are indicated.
(C) Immunoblotting to confirm the expression and integration of Venus tagged uS13 and uL5 onto ribosomes is shown. These were probed with an anti-GFP antibody. The upper and lower bound range of the protein marker with respect to Venus tagged uS13 (31 kDa) and uL5 (34 kDa) is shown. Lanes corresponding to ribosomes from WT and SLVNC are indicated. The ratio of the fraction of Venus tagged ribosomal proteins uS13 and uL5 (denoted as T) to Venus untagged uS13 and uL5 (denoted as U) is indicated at the bottom of each lane.
(D) Fluorescence complementation is shown for crude ribosomes purified from Wt, SLV and VNC strains. Lysate from VNC and SLVNC showed comparable fluorescence (inset).
(E) Fluorescence emissions from cell lysates of SVN, LVC, VNC, SVNC and LVNC
strains relative to lysate from SLVNC strain were measured (vide. text).
(F) Relative fluorescence intensities from LVNC and SLVNC ribosomesare shown after the crude ribosomes were treated with EDTA, RNase A and buffer
containing 1mM Mg2+ to dissociate the subunits. The fluorescence intensity normalized with respect to untreated ribosomes from SLVNC is shown.
In order to further reinforce that the BiFC signal from the purified crude ribosomes is due to the association of the 30S and 50S, we destabilized the subunit association by the addition of EDTA and RNase A. This led to the reduction in the fluorescence intensity significantly (Fig.3.2F). EDTA chelates the Mg2+ ions essential for subunit association and RNase A compromises the integrity of rRNA – both leading to the dissociation of the 30S and 50S to a varying extent – that was proficiently captured by the fluorescence complementation (Fig. 3.2F). A similar test was performed under low Mg2+ concentration that favors the dissociation of subunits. Here, the crude ribosomes were first purified and dialyzed against buffer containing 1 mM Mg2+. This showed a reduction in the BiFC signal (Fig. 3.2F). This suggests that indeed BiFC is highly sensitive and correlates well with the dissociation of the subunits.
Having reduced the background noise and confirmed the subunit association, we set out to ask whether the presence of tagged uS13 and uL5 alleles would impair the assembly. Therefore, we performed sedimentation analysis for the ribosomes prepared from the cell lysates of SLVNC and Wt under conditions that favor association of ribosomal subunits (10 mM Mg2+). For Wt, we observed majorly 70S and polysomes with minor amounts of 30S and 50S indicating that the association between 30S and 50S is robust.
The ribosome profile for SLVNC was similar to that of Wt suggesting that the assembly is not impaired by the presence of Venus tag on uS13 and uL5 (Fig. 3.3A & 3.3B).
Further, the BiFC measured from the fractionated crude ribosomes correlated well with the sedimentation profile displaying significantly high fluorescence signal for fractions corresponding to 70S and polysomes than those related to 30S and 50S. This suggests that BiFC indeed captures the association of the subunits (Fig. 3.3C). It is worthy to note that when the sedimentation analysis of the ribosomes was performed by loading the cell lysates directly onto the sucrose gradient, noticeable fluorescence corresponding to 30S and 50S fractions was observed (data not shown). This might have stemmed from the co-sedimentation of trace amounts of free Venus-tagged uS13 and uL5 along with the 30S and 50S subunits. In line with our earlier observation, however, when we separately purify crude ribosomes and then subjected them to sedimentation analysis, this background noise is largely alleviated (Fig. 3.3C).
Figure 3.3: BiFC profiles correlate with conventional ribosome profiles
(A) Ribosome profile for Wt is presented. The peaks pertaining to 30S, 50S, and 70S are indicated.
(B) Ribosome profile for SLVNC is presented.
(C) The BiFC measured from the corresponding fractions (in A) is shown for SLVNC. The BiFC profile is in agreement with the sedimentation profile of the ribosomes.
(D) An immunoblot is shown for equal amounts of ribosomes isolated from SLVNC
andSLVNC ΔrpsM cells. These were probed with the anti-GFP antibody. The upper and lower bound range of the protein marker with respect to Venus tagged uS13 (34.4 kDa) and uL5 (31.4 kDa) is shown (Left). This relative fluorescence intensity of crude ribosomes prepared from SLVNC ΔrpsM that was normalized with respect to the crude ribosomes from SLVNC (Right).
(E) Relative fluorescence intensities for crude ribosomes from SLVNC ΔrpsM that was normalized with respect to the crude ribosomes from SLVNC.
(F) The ribosome profile obtained from SLVNC ΔrpsM cell lysates. The peaks corresponding to 30S, 50S, and 70S are indicated.
In addition, we also created a null mutant of rpsM that codes for the non-essential S13 (Bubunenko et al., 2007) against the SLVNC background (SLVNC ΔrpsM). In this modified strain, the Venus tagged uS13 allele remains as the only source for uS13 and that allowed us to assess whether the Venus tag on uS13 would impair the assembly. The integration of Venus tagged uS13 onto the ribosomes was confirmed and that it displayed significant fluorescence complementation (Fig. 3.3D). Further, based on the correspondence between the ribosome profile and the BiFC signal, we confirmed that the Venus tag doesn’t alter the assembly (Fig. 3.3E & 3.3F). Similar such efforts to create a null mutant of rplE that codes for the essential L5 (Korepanov et al., 2007; Shoji et al., 2011) didn’t fructify despite several meticulous attempts. Therefore, it wasn’t possible to
assess the effect of tag against the SLVNC ΔrplM background. However, based on SLVNC
and SLVNC ΔrpsM, we believe that it is unlikely that the tag on uL5 will impact the assembly significantly.
3.3.3 BiFC detects maturation defects that are prompted by the