Relaxosome

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The relaxosome is the complex of proteins that facilitates plasmid transfer during bacterial conjugation. The proteins are encoded by the tra operon on a fertility plasmid in the region near the origin of transfer, oriT. The most important of these proteins is relaxase, which is responsible for beginning the conjugation process by cutting at the nic site via transesterification. This nicking results in a DNA-Protein complex with the relaxosome bound to a single strand of the plasmid DNA and an exposed 3' hydroxyl group. Relaxase also unwinds the plasmid being conjugated with its helicase properties. The relaxosome interacts with integration host factors within the oriT.

Plasmid small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently

A plasmid is a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism, such as by providing antibiotic resistance. While the chromosomes are big and contain all the essential genetic information for living under normal conditions, plasmids usually are very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation.

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus.

An origin of transfer (oriT) is a short sequence that is necessary for transfer of the DNA that contains it from a bacterial host to recipient during bacterial conjugation. The oriT is cis-acting — it is found on the same DNA that is being transferred, and it is transferred along with the DNA. The origin of transfer consists of three functionally defined domains: a nicking domain, a transfer domain, and a termination domain.

Other genes that code for relaxosome components include TraH, which stabilizes the relaxosome's structural formation, TraI, which encodes for the relaxase protein, TraJ, which recruits the complex to the oriT site, TraK, which increases the 'nicked' state of the target plasmid, and TraY, which imparts single-stranded DNA character on the oriT site. TraM plays a particularly important role in relaxase interaction by stimulating 'relaxed' DNA formation.

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Pilus

A pilus is a hair-like appendage found on the surface of many bacteria. The terms pilus and fimbria can be used interchangeably, although some researchers reserve the term pilus for the appendage required for bacterial conjugation. All pili in the latter sense are primarily composed of pilin proteins, which are oligomeric.

Hfr cell hfr-strain


A high-frequency recombination cell is a bacterium with a conjugative plasmid integrated into its chromosomal DNA. The integration of the plasmid into the cell's chromosome is through homologous recombination. A conjugative plasmid capable of chromosome integration is also called an episome. When conjugation occurs, Hfr cells are very efficient in delivering chromosomal genes of the cell into recipient F cells, which lack the episome.

The origin of replication is a particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses.

<i>Agrobacterium tumefaciens</i> species of bacterium

Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA, from a plasmid, into the plant cell, which is incorporated at a semi-random location into the plant genome.

Ti plasmid

A Ti or tumour inducing plasmid is a plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce their genetic material to plants. The Ti plasmid is lost when Agrobacterium is grown above 28 °C. Such cured bacteria do not induce crown galls, i.e. they become avirulent. pTi and pRi share little sequence homology but are functionally rather similar. The Ti plasmids are classified into different types based on the type of opine produced by their genes. The different opines specified by pTi are octopine, nopaline, succinamopine and leucinopine.

The fertility factor allows genes to be transferred from one bacterium carrying the factor to another bacterium lacking the factor by conjugation. The F factor is carried on the F episome, the first episome to be discovered. Unlike other plasmids, F factor is constitutive for transfer proteins due to a mutation in the gene finO. The F plasmid belongs to a class of conjugative plasmids that control sexual functions of bacteria with a fertility inhibition (Fin) system.

Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.

A relaxase is a single-strand DNA transesterase enzyme produced by some prokaryotes and viruses. Relaxases are responsible for site- and strand-specific nicks in double-stranded DNA. Known relaxases belong to the Rolling Circle Replication (RCR) initiator superfamily of enzymes and fall into two broad classes: replicative (Rep) and mobilization (Mob). The nicks produced by Rep relaxases initiate plasmid or virus RCR. Mob relaxases nick at origin of transfer (oriT) to initiate the process of DNA mobilization and transfer known as bacterial conjugation. Relaxases are so named because the single-stranded DNA nick that they catalyze lead to relaxation of helical tension.

fis is an E. coli gene encoding the Fis protein. The regulation of this gene is more complex than most other genes in the E. coli genome, as Fis is an important protein which regulates expression of other genes. It is supposed that fis is regulated by H-NS, IHF and CRP. It also regulates its own expression (autoregulation). Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions.

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed binary vectors because of their ability to replicate in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a bacterium used to insert the recombinant (customized) DNA into plants. Plant Transformation vectors contain three key elements;

The nic site or nick region is found within the origin of transfer (oriT) site and is a key in starting bacterial conjugation.[1]

The traA gene codes for relaxase, which is an enzyme that initiates plasmid DNA transfer during bacterial conjugation. Relaxase forms a relaxosome complex with auxiliary proteins to initiate conjugation. Relaxosome binds to the origin of transfer (oriT) sequence and cleaves the DNA strand that will be transferred.

Transfer operon, commonly called tra operon, or tra genes, are some genes necessary for non-sexual transfer of genetic material in both gram-positive and gram-negative bacteria. The tra locus includes the pilin gene and regulatory genes, which together form pili on the cell surface, polymeric proteins that can attach themselves to the surface of F-bacteria and initiate the conjugation. The existence of the tra region of a plasmid genome was first discovered in 1979 by David H. Figurski and Donald R. Helinski In the course of their work, Figurski and Helinski also discovered a second key fact about the tra region – that it can act in trans to the mobilization marker which it affects.

In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

TraJ-II RNA motif

The traJ-II RNA motif is a conserved RNA structure discovered in bacteria by using bioinformatics. traJ-II RNAs appear to be in the 5' untranslated regions of protein-coding genes called traJ, which functions in the process of bacterial conjugation. A previously identified motif known as TraJ 5' UTR is also found upstream of traJ genes functions as the target of FinP antisense RNAs, so it is possible that traJ-II RNAs play a similar role as targets of an antisense RNA. However, some sequence features within the traJ-II RNA motif suggest that the biological RNA might be transcribed from the reverse-complement strand. Thus is it unclear whether traJ-II function as cis-regulatory elements. traJ-II RNAs are found in a variety of proteobacteria.

Bacterial genetics is the subfield of genetics devoted to the study of bacteria. Bacterial genetics are subtly different from eukaryotic genetics, however bacteria still serve as a good model for animal genetic studies. One of the major distinctions between bacterial and eukaryotic genetics stems from the bacteria's lack of membrane-bound organelles, necessitating protein synthesis occur in the cytoplasm.

Plasmids must regulate their copy number to ensure that they do not excessively burden the host or become lost during cell division. Plasmids may be either high copy number plasmids or low copy number plasmids; the regulation mechanisms between these two types are often significantly different. Biotechnology applications may involve engineering plasmids to allow a very high copy number. For example, pBR322 is a low copy number plasmid from which several very high copy number cloning vectors have been derived.

References

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