The Role Of Acetylation To Ribosomal Protein RplB And Characterization Of The Deacetylase And Auto-ADP-Ribosyltransferase MSMEG₄620

PART 1: THE ROLE OF ACETYLATION TO RIBOSOMALPROTEIN RPLBThe role of acetylation have been broadly and deeply studied in eukaryotes, but in prokaryotes, although a large number of proteins have been reported to be acetylated, involved in almost all the life processes, such as energy metabolism, RNA transcription, chemotaxis and so on, the effect of acetylation on the function remains poorly understood. In this part, acetyltransferase Pat was purifiedand combined with the acetylation reaction we have found that ribosomal protein Rpl B can be acetylated by Pat; Rpl B, which interacts with replication initiation protein Dna A, RNA polymerase and can also bind with r RNA, is an indispensable part for protein translation. In order to further investigate the role of acetylation to ribosomal protein Rpl B, in this part we adopts the means of molecular biology, microbiology and biochemistry in E. coli K12 strain BW25113 to expand the study into the following parts:In the first, we found that Rpl B can interact with Pat and Cob B through GSTpulldown method, and then we proved Rpl B can be directly acetylated by Pat and deacetyed by Cob B; Because of that Rpl B binds to r RNA depends on the positive charge on the protein and acetylation will neutralize the positive charge of lysine, so in order to investigate the role of acetylation on Rpl B, acetylated and non-acetylated Rpl B was incubated with r RNA respectively and we found that acetylation significantly decreased the binding ability of Rpl B with r RNA through agarose gel electrophoresis. Following that, the lysine content in ribosomal and non ribosomal proteins were analyzed and we found that the content of the former is significantly higher than the latter, suggesting that acetylation may affect ribosome assembly and translation efficiency, which is consistent with that cob B knockout strain grows slower than WT.In the second, to study whether Rpl B can be acetylation-regulated by Pat / Cob B in vivo, we constructed a plasmid p SUMO-rpl B and co-expressed with p ET28a-cob B or p ET28a-pat in BL21, we found that acetylation of Rpl B decreased when co-expressed with Cob B while acetylation increased when co-expressed with Pat, indicating Pat and Cob B regulated in vivo acetylation of Rpl B; However acetylation of Rpl B is not the same when expressed in BW25113 and pat, cob B knockout strains, in cob B knockout strain acetylation of Rpl B was significantly higher than WT, while acetylation didn’t change in pat knockout strain, it suggests there are other factors to modify Rpl B; Recently it’s reported that Ac P can be used as donor to acetylate proteins, when Rpl B is treated with Ac P, we found acetylation of Rpl B significantly increased.In the third, in order to investigate whether acetylation of Rpl B changes under the condition of nitrogen starvation, we transferred bacteria in log phase into nitrogen deficient PBS plus 0.5% glucose as starvation treatment and found that with time, acetylation of Rpl B gradually increased.In summary, ribosomal protein Rpl B can be acetylation-regulated by acetyltransferase Pat and deacetylase Cob B, and acetylation will reduces its binding ability with r RNA; in addition, we found acetylation of Rpl B gradually increased under the condition of nitrogen starvation and lysine content of ribosomal proteins were significantly higher than non-ribosomal proteins, suggesting that in the nitrogen starvation conditions, bacteria can adjust the acetylation of Rpl B or the whole ribosome to regulate its own growth rate. These results have advanced the research about the role of acetylation on the function of proteins, and opened up a new field of the ribosome. PART 2: CHARACTERIZATION OF THE DEACETYLASE AND AUTO-ADP-RIBOSYLTRANSFERASE MSMEG₄620Sir2 family proteins are highly conserved proteins in structure and function, involved in almost all the life processes including gene silencing, DNA damage repair, growth and metabolism; Here, in addition to the presence of Sir T2 homologue protein MSMEG₅175, we have discovered a SIRT4 homologue protein MSMEG₄620 in Mycobacterium smegmatis mc2155 through NCBI Blast, and then the distribution of SIRT2 and SIRT4 homologue proteins in the various strains of mycobacteria is analyzed, we found that the former contains only a SIRT2 homologue protein while the latter contains two homologue proteins, indicating that SIRT4 homologue protein are closely related with nutrition and primary metabolism. In this part we adopts the means of molecular biology, microbiology and biochemistry to expand the study into the following parts:In the first, through extending the homology arms much longer we constructed a MSMEG₄620 knockout strain of mc2155, and then we compared the difference of growth rate with WT strain in the rich or barren medium; In the rich medium, we found it shows no difference in growth between them, while in the poor medium, MSMEG₄620 knockout strain grows much slower than wild type strain, these results suggest that MSMEG₄620 plays a very important role in the strain which grows in wild.In the second, to study the deacetylation activity MSMEG₄620, we applied a chemical substrate by measuring fluorescence and the cytosolic proteins by Western blot, we found that MSMEG₄620 has weak deacetylation activity. But knockout of MSMEG₄620 significantly increased acetylation of cytosolic proteins, suggesting that MSMEG₄620 in vivo can be activated; We tried to add various fatty acids into in vitro reaction and found that the oleic acid can activate the deacetylation activity of MSMEG₄620.In the third, to investigate the ADP-ribosyltransferase activity of MSMEG₄620, we applied biotin-NAD+ into the reaction and used avidin-HRP for Western blot to measure the ADP-ribosyltransferase activity. The results showed that MSMEG₄620 can ADPribosylate itself other than the histone, and the activity can not be inhibited by ADP-ribose while free ADP-ribose can not be detected by HPLC method, these all suggest it is not a NADase. We then performed the mutations of the conserved amino acids, most of which show significantly decreased ADP-ribosyltransferase activity; In the next, followed by chemical treatment, we found that NH2 OH can remove the modified ADP-ribose goup on MSMEG₄620 illustrating it is modified on arginine. Finally, according to the recent report, a variety of metal ions were added in the reaction respectively, we found Fe3 + can enhance ADP-ribosyltransferase activity of MSMEG₄620.In the fourth, the deacetylation activity of ADP-ribosylated and unmodified MSMEG₄620 was compared and it shows no significant difference, indicating the ADPribosylation of MSMEG₄620 doesn’t affect its deacetylation activity. No matter it shows which kind of activity, MSMEG₄620 will consume NAD+, so we compared the NAD+ levels between MSMEG₄620 knockout strain and WT, and found that the former is higher than the latter.In summary, we have firstly found MSMEG₄620 shows fatty acid-activated deacetylation activity and Fe³⁺ activated auto-ADP-ribosyltransferase activity, both coregulates the NAD+ level in vivo; In addition, MSMEG₄620 likely plays an important role in relatively poor medium, after knockout of MSMEG₄620, bacteria shows significantly decreased growth rate relative to WT. These findings will advance the research of MSMEG₄620 and Sir2 family proteins in prokaryotes, especially the physiological functions.