Biomedical subjects

Megan E Rokop

Publications and source records attributed to Megan E Rokop.

3 recordsLinked to original sources

PubMed2004

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes.

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.

3' Untranslated Regions↗

PubMed2004

Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis.

The Bacillus subtilis proteins DnaD and DnaB are essential for replication initiation and are conserved in low G+C content Gram-positive bacteria. Previous work indicated that DnaD and DnaB are involved in helicase loading during the process of restarting stalled replication forks. We have investigated the roles of DnaD and DnaB in replication initiation at oriC in vivo. Using chromatin immunoprecipitation (ChIP), we found that DnaD and DnaB functions are needed to load the replicative helicase at oriC. To investigate further the functions of DnaD and DnaB in replication initiation, we isolated and characterized suppressors of the temperature sensitivity of dnaD and dnaB mutant cells. In both cases, we isolated the identical missense mutation in dnaB, dnaBS371P. Using yeast two-hybrid analysis, we found that dnaBS371P uncovers a previously undetected physical interaction between DnaD and DnaB. We also found that DnaBS371P constitutively recruits DnaD to the membrane fraction of cells, where DnaB and oriC are enriched. Phenotypes of cells expressing DnaBS371P are consistent with aberrant replication control. We hypothesize that B. subtilis regulates replication initiation by regulating a physical interaction between two proteins essential for helicase loading at chromosomal origins.

Bacillus subtilis↗

PubMed2004

Multicopy plasmids affect replisome positioning in Bacillus subtilis.

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

Bacillus subtilis↗