Identification of Potential Monomeric Transcription Factors in Mycobacterium tuberculosis Through Sequence Analysis

Camden Jones Author

04/05/2021 Added

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This video presents research done on the identification of potential monomeric transcription factors found in mycobacterium tuberculosis through methods of sequence analysis.

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[00:00:01.170]Hello, my name is Camden Jones.
[00:00:03.880]My mentor on this project project was Dr. Limei Zhang
[00:00:06.620]and I received additional guidance from my associates
[00:00:08.820]Magdalena Horova and Daisy Guiza Beltran.
[00:00:12.030]I will be presenting my project from this academic term
[00:00:14.840]on the Identification
[00:00:16.300]of Potential Monomeric Transcription Factors
[00:00:19.150]in Mycobacterium tuberculosis Through Sequence Analysis.
[00:00:23.430]Mycobacterium tuberculosis or Mtb
[00:00:26.070]is the causative agent of the disease tuberculosis.
[00:00:29.550]This pathogen uses a multitude of genes
[00:00:31.680]that regulate the complex mechanisms
[00:00:33.410]used to promote virulence.
[00:00:35.400]Virulence is a pathogens ability to cause damage
[00:00:37.450]in a host.
[00:00:38.850]Transcriptional regulation controls the expression
[00:00:41.050]of the genes involved in these mechanisms.
[00:00:43.690]Which allows Mtb to adapt to stress environments
[00:00:46.610]created by the host.
[00:00:48.530]These transcriptional regulators
[00:00:50.330]are known as transcription factors.
[00:00:52.400]Transcription factors are proteins that regulate
[00:00:54.750]gene transcription by binding to DNA.
[00:00:58.700]Transcription factors can be characterized
[00:01:01.010]based on their oligomeric state.
[00:01:03.160]This study is specifically interested
[00:01:04.880]in monomeric transcription factors found in Mtb.
[00:01:08.500]Monomeric transcription factors are intriguing
[00:01:10.800]because there are not well characterized
[00:01:12.370]and there are a lot of unknowns
[00:01:13.530]when it comes to understanding its functions
[00:01:15.300]and importance in transcription regulation
[00:01:17.940]in pathogens such as Mtb.
[00:01:21.310]Monomeric transcription factors consists
[00:01:23.190]of one protein that does not bind
[00:01:24.810]to other transcription factors
[00:01:26.230]in order to bind to DNA and regulate transcription.
[00:01:29.230]However, monomeric transcription factors
[00:01:31.370]are able to bind to other proteins like sigma factors
[00:01:34.210]and still retain their monomeric function.
[00:01:37.190]The most common transcription factors in Mtb are dimeric.
[00:01:40.940]The differences between these oligomeric States
[00:01:42.930]are highlighted in figure one.
[00:01:45.180]The transcription factors analyzed were used
[00:01:47.660]from a list provided by the 2016 Feng study,
[00:01:50.820]which presented potential monomeric transcription factors
[00:01:54.080]in Mtb from a list of 154 transcription factors.
[00:01:59.990]The visual representation given here is that of figure one.
[00:02:02.830]This figure provides a visual representation
[00:02:04.910]of the difference between monomeric transcription factors
[00:02:07.087]and dimeric transcription factors.
[00:02:09.630]This figure uses MogR as the example
[00:02:11.540]for showing this difference.
[00:02:13.550]As can be seen in the first part of the figure,
[00:02:16.200]when MogR is functioning as a monomer,
[00:02:18.920]it does not interact with other transcription factors
[00:02:21.450]such as another MogR transcription factor.
[00:02:24.670]The figure shows that in this monomeric state,
[00:02:26.730]MogR is not able to bind into its binding site.
[00:02:30.210]However, when MogR is functioning as a dimer,
[00:02:34.050]it binds with another MogR transcription factor
[00:02:36.420]shown here in this complex.
[00:02:39.020]In this form, the MogR is able to bind
[00:02:41.300]to the specific binding site
[00:02:44.120]and reduce transcription
[00:02:45.840]by interacting with the Sigma region of RNAP.
[00:02:48.960]And then this detaches it from its binding site.
[00:02:53.750]Just to emphasize the point of this study.
[00:02:56.240]The purpose to identify and study unique monomeric transcription factors
[00:03:00.040]in Mtb via sequence analysis.
[00:03:03.470]The end goal of this research is to gain an understanding
[00:03:05.750]behind the function of monomeric transcription factors
[00:03:08.890]within the regulatory systems of Mtb.
[00:03:11.540]That contributes to the pathogens
[00:03:13.020]high tolerance to host stress environments and antibiotics.
[00:03:16.640]More understanding that is obtained
[00:03:18.080]about monomeric transcription factors,
[00:03:20.010]the closer we can get to producing new drugs to treat Mtb.
[00:03:24.710]The main methods used in this research
[00:03:26.390]to identify the oligomeric state,
[00:03:28.250]were methods of sequence analysis.
[00:03:30.570]By using these methods,
[00:03:31.920]we were able to get a closer look
[00:03:33.220]at the sequence of the target transcription factor
[00:03:35.560]and get more information on its possible function.
[00:03:38.570]Databases such as BLAST and Mycobrowser,
[00:03:40.770]were used for most of the sequence analysis.
[00:03:43.290]The data generated from BLAST search was used to analyze
[00:03:45.920]the possible function of the transcription factors.
[00:03:48.110]Since most of the ones provided
[00:03:49.640]in Feng's list did not have known functions.
[00:03:53.490]This is shown in figure two.
[00:03:55.535]As can be seen in the figure,
[00:03:57.410]the main information that was able to be found
[00:03:59.360]from these databases were the gene name,
[00:04:02.210]the locus tag
[00:04:03.840]the possible function
[00:04:05.410]and comments that were found based on BLAST searches.
[00:04:10.040]When we look at this column where it says
[00:04:11.640]predicted to be monomeric,
[00:04:13.003]this is based on what was found in the Feng study.
[00:04:15.790]For example, for the gene Rv zero, five, five, two.
[00:04:19.640]Feng and his associates propose that this was monomeric.
[00:04:25.280]Mycobrowser provides the most recent information
[00:04:27.900]on these transcription factors in Mtb.
[00:04:30.550]This information
[00:04:31.383]can help elucidate a putative function as well.
[00:04:34.200]And this is shown as an example in figure three.
[00:04:37.240]The upper half of the figure shows where
[00:04:38.980]in the genomic sequence of Mtb the gene is located.
[00:04:42.520]This information is helpful to understand
[00:04:44.220]how the gene may function based on its location
[00:04:46.500]in relation to other genes in the sequence.
[00:04:48.570]The lower half of the figure
[00:04:50.510]shows more information about the particular gene.
[00:04:53.390]For Rv zero five, five, two,
[00:04:55.690]Mycobrowser was able to provide more information
[00:04:57.960]on its location,
[00:04:59.360]like how it is upstream from the transcription factor
[00:05:03.010]M-E-N-C in a possible function.
[00:05:07.030]In this case, the possible functions
[00:05:08.900]that have an amidohydrolase.
[00:05:12.460]After this initial analysis
[00:05:14.080]those transcription factors
[00:05:15.610]that were found to be possibly monomeric
[00:05:17.670]were kept in a new list,
[00:05:19.090]while the rest of the genes from the list were removed.
[00:05:22.770]After collecting data from these databases,
[00:05:25.480]we needed a form of analysis.
[00:05:27.750]SWISS-MODEL and Chimera
[00:05:28.870]were the programs use to analyze the data.
[00:05:31.560]By building possible models for each gene
[00:05:33.660]in the narrowed list through SWISS-MODEL,
[00:05:35.960]identification of the oligomeric state of the gene
[00:05:38.120]is possible.
[00:05:39.294]Chimera is then used to analyze the models oligomeric state
[00:05:42.030]by giving a closer look into the interactions
[00:05:44.390]within a protein and this is shown in figure four.
[00:05:47.660]In this figure, the target transcription factor
[00:05:49.740]which is highlighted here in blue.
[00:05:51.540]Is found to be part of a larger complex
[00:05:53.390]that consists of a subset of eight proteins.
[00:05:55.470]And that is the whole thing here.
[00:05:57.590]This entire complex is that of a toxin antitoxin system
[00:06:01.100]called the vapBC11 complex.
[00:06:04.410]By analyzing the contacts
[00:06:05.960]which is shown in these yellow lines
[00:06:08.090]between the target transcription factor
[00:06:09.730]and the rest of the protein,
[00:06:11.150]I found this protein to be monomeric.
[00:06:13.910]By comparing the target sequence
[00:06:15.620]and the sequence from the model.
[00:06:17.550]It was found that some essential hydrophobic residues
[00:06:20.110]that promote dimerization were not conserved
[00:06:22.330]while others were.
[00:06:23.980]Since this entire complex functions
[00:06:25.780]as a toxin anti-toxin system,
[00:06:28.300]this gene acts as a monomeric transcription factor
[00:06:30.570]within the complex.
[00:06:32.300]This is indicated by the conserved residues
[00:06:34.290]and the possible function of the gene.
[00:06:37.370]Through this work,
[00:06:38.203]I found that of the 154 transcription factors
[00:06:41.030]in Feng study 13 of them
[00:06:42.450]present to be possible monomeric transcription factors.
[00:06:45.420]These are intriguing results
[00:06:46.440]considering things study proposed there
[00:06:47.920]to be 32 possible monomeric transcription factors.
[00:06:51.120]These differing findings could be the result
[00:06:52.830]of there being more information known
[00:06:54.240]about the transcription factors found in Mtb
[00:06:56.360]than when the study was published by Feng
[00:06:57.950]and their associates.
[00:07:00.350]It was also possible that there was not enough
[00:07:02.360]supporting evidence to make a conclusive assertion
[00:07:04.920]on oligomeric state of some of the transcription factors.
[00:07:07.760]Especially if there is not as much available information
[00:07:10.040]at the time of publication.
[00:07:12.170]These findings convey how much is not known
[00:07:13.940]about monomeric transcription factors
[00:07:15.640]and their importance in transcriptional regulation in Mtb.
[00:07:18.730]This result leads me to the future work
[00:07:21.100]I will be performing next in the Dr.Zhang research lab.
[00:07:24.490]The future work to be done
[00:07:25.550]in order to provide evidence to confirm or deny
[00:07:27.740]whether the new list to propose
[00:07:29.070]monomeric transcription factors are indeed monomeric,
[00:07:32.510]includes laboratory techniques
[00:07:34.050]such as PCR cloning, protein overexpression
[00:07:37.660]pull-down assays, GFP flourescence emission spectroscopy
[00:07:41.600]and sodium dodecyl sulfate
[00:07:43.092]polyacrylamide gel electrophoresis,
[00:07:45.690]or SDS-PAGE analysis.
[00:07:48.750]My references are provided in the section below.
[00:07:51.930]I would like to give special thanks
[00:07:53.430]to UCARE in sponsoring this research
[00:07:55.720]as well as Dr. Zhang and her associates
[00:07:57.560]within the Dr. Zhang lab
[00:07:58.880]that helped make this research possible.
[00:08:01.020]Thank you for your time.

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Identification of Potential Monomeric Transcription Factors in Mycobacterium tuberculosis Through Sequence Analysis

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