Transposable Elements and Transposons

These chromosomal loci show the ability to be transposed from one location to another, within and among the chromosomes of the compliment. Breaks on either side of the loci followed by insertion of the broken transposon into a new locus are known as transposition. Hence, a transposable element gets inserted into a chromosome, and also exists or relocates itself. A group of transposable elements includes insertion sequences, transposons, retrotransposons, bacteriophages, and P elements. The inverted repeats flank the region of transposable elements or the transposons. These mobile genetic elements are also known as jumping genes. These elements also belong to the class of junk DNA.

Transposable elements in prokaryotes:

·        Insertion sequences:

It consists of only those genes which require mobilization and get inserted in a new location in a chromosome. The IS elements are present in the bacterial chromosomes and the plasmids. The studies related to bacterial IS elements and effect of the expression of the genes controlling the galactose metabolism revealed an insertion of 800 base pair DNA segment in the gene. It is known as the insertion sequence-1 involving the transposition. The IS elements get integrated into the chromosome randomly. Hence, such changes involve very high chances of the disruption of the coding sequences or the regulatory sequences. The crossing over between the segments having the IS elements lead to deletions or inversions. The transposition process requires an enzyme known as a transposase. This enzyme recognizes the IS elements and helps in initiating the transposition.

The process of insertion occurs at a target site. It leads to the duplication of the target site leading to the formation of the direct target repeats. Now, the IS element exactly places itself between the two inverted repeats flanked by direct target-site duplications. The gaps get filled by DNA polymerase and ligase enzymes.

·      Transposon:

It is a complex structure consisting of the genes required for the insertion and mobilization of the DNA into the chromosome. The two types of transposons include composite and the non-composite transposons. The composite transposons have a complex structure. The central region consists of genes. This region gets flanked by IS elements on both the sides. The non-composite transposons include drug resistance genes. A composite transposon (for example Tn10) consists of a central region carrying a gene flanked by IS elements having inverted repeats. It also gets flanked by direct sites. The example of non-composite transposon includes Tn3. It consists of genes for three main enzymes in the central part. The bla gene encodes the beta-lactamase enzyme. The tnpA gene encodes transposase enzyme. The TnpB encodes resolvase enzyme.

Image 1: IS elements

Eukaryotic transposable elements:

Plants include several families of transposons. The transposons consist of autonomous and non-autonomous elements. The autonomous elements transpose by themselves since they lack the gene for transposition. The derivative of the autonomous element having loss of function of one or few genes is known as the nonautonomous element. The insertion of the autonomous element into the host gene makes the mutant allele unstable. Some of the corn kernels show the presence of spots. These spots arise due to the pigments produced by the cells having a transposable genetic element.

The corn consists of a pigment known as anthocyanin. It gives the corn kernel purple color. Mutation in the gene responsible for the production of anthocyanin leads to an unpigmented kernel. The corn kernel having a wild-type C gene gives a purple color. The recessive c gene gives colorless kernels. These mutations block the production of anthocyanin. The revertants of mutations give purple spots on the kernels. The colorless mutation arises due to the mobile controlling element. These mobile controlling elements are known as Ds depicting the dissociation. They get inserted into the C gene (wild-type). One more mobile controlling element, known as Ac is known as an activator. It helps in the transposition of Ds into the gene.

The Ac-Ds transposable elements in corn included studies carried out by Barbara McClintock. The cut and paste transposition of the Ac element occurs during the chromosome replication. Upon replication of the chromosomal region containing Ac site, each progeny chromatid gets a copy of Ac. The transposition of the Ac element to a replicated site on the chromosome results in the empty donor site on one chromatid and Ac element on the other chromatid. The Ac elements do not increase in number if they get inserted into an already replicated site. Consider another case in which the Ac transposition occurs in an unreplicated chromosome site. First, one of the chromatids gets an empty donor site. The other chromatid consists of the Ac element. In this case, the transposing element gets inserted into the nearby recipient site ready to be replicated. The result of replication involves the presence of the Ac element on both the chromatids. Hence, this type of transposition leads to an increase in the number of Ac elements. The transposition of the Ds elements occurs in the same way.

Image 2: Cointegration model

Ty elements in yeast:

These elements include long terminal repeats. The yeast Ty elements consist of a length of 5.9 kb. The long terminal repeats are known as directly repeated terminal sequences. The denotion of long terminal repeats involves a delta sign (δ). Each delta consists of a promoter region and sequences recognized by transposing enzymes. The Ty elements encode a single mRNA having promoter elements at the delta region. Open reading frames in the mRNA transcript encode different proteins. They include TyA and TyB open reading frames. The Ty elements transpose by making an RNA molecule of the integrated DNA sequence. Hence, it creates a new Ty element by the process of reverse transcription. The yeast Ty elements follow the same mechanism conducted by retroviruses. They replicate via double-stranded DNA.

Drosophila transposons:
The mobile genetic elements in Drosophila constitute about 15% of the total genome content. The example of Drosophila transposons includes P element. These elements have terminal inverted repeats. The autonomous P elements are the longest ones. The nonautonomous elements include the shortest ones. The autonomous P elements encode for transposase enzyme. This enzyme helps in the transposition of the P elements. The P elements serve as vectors for transferring the genes.

LINEs and SINEs:

The human retrotransposons consist of LINES and SINES. Long interspersed sequences (LINEs) and short interspersed sequences (SINEs) occur in the moderately repetitive class of sequences. LINEs include autonomous elements that encode enzymes for the retrotransposition. The nonautonomous elements or SINEs also require those enzymes. The example of LINEs includes an L1 element with a size of 6500 base pair. The example of SINEs includes Alu family.

References:
[1] Biotechnology-3: Including Molecular Biology Biophysics, S. Mahesh
[2] Transposable element - Wikipedia

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Translation in prokaryotes

The process of protein synthesis from a mRNA molecule is known as translation. It occurs mainly in the ribosomes. Consider the messenger RNA structure like a tape. As soon it attaches to the ribosome, the process of protein synthesis starts. The movement of the RNA molecule starts creating a lengthy polypeptide chain. First, the leading end on mRNA (the 5' end) emerges from the first ribosome. Next, it attaches to the second ribosome for synthesizing the next polypeptide chain. Once the RNA moves from a ribosome, it gets vacated for the new set of instructions. The amino acids start getting assembled into a polypeptide chain from the amino terminus. The process of assembly finishes at the carboxylate terminus. During the process of translation, an RNA molecule passes on the amino acid molecule to the growing polypeptide chain. Hence, it is known as a tRNA molecule. The transfer RNAs belong to a class of smallest biologically active molecules. The transfer RNAs attach to the amino acids at their 3' end. 

The translation of mRNA occurs from 5’ to 3’ direction. An amino acid binds to its specific tRNA. The codon of mRNA binds to the anticodon of tRNA through a complementary base pairing. The mRNA is specific in recognizing the anticodon. An enzyme known as aminoacyl tRNA synthetase attaches a correct amino acid to the tRNA. This process is known as aminoacylation or charging. Let us consider the example of valine tRNA. The first step involves binding of amino acid and ATP to a specific aminoacyl tRNA synthetase. Hence, the reaction leads to the loss of two phosphate groups from ATP. It leads to the formation of aminoacyl AMP. The next step involves binding of uncharged tRNA to the enzyme. As a result, the enzyme transfers the amino acid to tRNA. Hence, the aminoacyl tRNA releases from the enzyme. A factor known as transfer factor I participates in binding the charged tRNA to the ribosome. The transfer factor II is also known as translocase. It is a protein capable of forming a complex with the GTP and the ribosome. When the charged tRNA gets translocated from its ribosomal entrance site to the peptidyl site, it leads to the hydrolysis of GTP to GDP, thereby releasing the translocase.

Image 1: Translation initiation and peptide bond formation: (1) The first image depicts the binding of the fMet tRNA to the initiation site. (2) The serine tRNA binds in the A site. (3) Peptide bond formation (4) The uncharged tRNA moves to the E site and the tRNA consisting of amino acids linked through peptide bond moves to the P site.

Initiation of translation:

The mRNA with an AUG initiation codon gets involved in the process of translation. The mRNA ribosome binding site (also known as Shine Dalgarno sequence) also contributes to the process of translation. The 30S ribosomal subunit binds to AUG start codon. Later on, the initiator tRNA binds to this site. Since, AUG start codon codes for methionine amino acid, the newly made proteins start with amino acid methionine. In prokaryotes, the methionine gets modified to formylmethionine (fMet). The fMet tRNA has a 5’-CAU-3’ anticodon. Binding of fMet tRNA to the start codon releases an IF3. Hence, it forms a 30S initiation complex. It consists of mRNA, 30S subunit, fMet tRNA, IF1, and IF2. The next step involves binding of the 50S ribosomal subunit, leading to the hydrolysis of GTP. This reaction releases the IF1 and IF2 factors. It finally leads to the formation of the 70S initiation complex. A site-P site hypothesis describes the ribosomal sites. Following are the three main binding sites for the aminoacyl tRNA:

·        The exit site is also known as the E site.

·        The peptidyl site is also known as the P site.

·        Aminoacyl site is also known as A site.

The fMet tRNA binds to the mRNA at the P site. A site accepts the incoming aminoacyl tRNA. For transferring the peptide group, it requires translocation of tRNA. Hence, the tRNA gets translocated from the A site to the P site.

Elongation:

The amino acids get added to the polypeptide chain thereby allowing it to grow till the required amount. It involves three main steps. Primarily, the aminoacyl tRNA comes in contact with the ribosome and binds to it. Secondly, it leads to the formation of the peptide bond through an enzymatic reaction. The third step involves the movement of the ribosome (translocation) along the mRNA. It considers one codon at a time. The peptidyl site of the ribosome comes in contact with the AUG codon. It is suitable for the fMet tRNA to bind to the mRNA. The tRNA anticodon helps in binding to the mRNA codon. Next step involves binding of the aminoacyl tRNA (for example ser tRNA) in the A site. Binding of an aminoacyl tRNA to the codon in the A site releases the elongation factor known as EF-Tu. The reaction involves the hydrolysis of GTP. Note that the elongation factors EF-Tu get recycled for the next aminoacyl tRNA.  Now, the fMet tRNA and aminoacyl tRNA come close to each other. Since these two amino acids are adjacent to each other, a peptide bond forms between the two amino acids. In this case, a peptide bond forms between the formylmethionine and serine amino acids. The reactions get catalyzed by peptidyl transferase.

The amino acids linked by a peptide bond get attached to the aminoacyl tRNA situated at the A site. The tRNA is now known as peptidyl tRNA. Next step involves the process of translocation. The ribosome moves from one codon to another codon. This step again involves EF-G factors and GTP. Hence, the peptidyl tRNA moves from A site to the P site, leaving the A site empty. Similarly, the uncharged tRNA moves to the E site from the P site. An uncharged tRNA is a tRNA that has given its amino acid for peptide chain elongation.

The tRNA without an attached amino acid gets released. Next, the ribosome starts preparing for the next elongation cycle. The empty A site gets occupied by another aminoacyl tRNA with a specific anticodon. The above process gets repeated till the sufficient proteins get synthesized.

Image 2: Elongation and termination of translation: (5) Next aminoacyl tRNA binds to the A site. (6)Peptide chain elongation. (7) Termination of the process due to the activity of the release factor. (8) Dissociation.

Termination:

None of the tRNAs possess anticodon for a stop codon. A protein group known as termination factor or release factor (RF) help the ribosome in recognizing a stop codon. There are three types of release factors in E. coli such as RF1, RF2, and RF3. Each RF is a single polypeptide. The role of RF1 involves recognition of UAA and UAG codons. The RF2 involves recognition of UAA and UGA. The RF3 does not recognize any stop codon. The ribosome recognizes a chain termination codon (UAG). Then the polypeptide chain present on the peptidyl tRNA (present on the P site) gets released. The ribosomal subunits get dissociated thereby separating the remaining components.

References:

[1] Principles of genetics, Gardner, M. J. Simmons, D. P. Snustad, eighth edition.

[2] Biology of the Prokaryotes, edited by Joseph W. Lengeler, Gerhart Drews.
[3] Genetics, G. Archunan
[4] Genetics, Daniel Hartl, Maryellen Ruvolo

© Copyright, 2018 All Rights Reserved.

Transcription in Prokaryotes

DNA is a special part of the cell. Without DNA, there would not have been life, evolution, survival, and existence. The base of synthesizing RNA starts from a DNA strand. Hence, DNA has a strict impact on the processes occurring in the body. DNA gets transcribed into RNA. Studying transcription helps to investigate how mutations affecting transcription cause inherited diseases. The first step involved in gene expression is transcription. Interested in cloning or treating a genetic disorder with DNA? Then keeping updates regarding transcription processes is helpful. RNA is a key nucleic acid involved in synthesizing proteins. Watson and Crick proposed the central dogma. It is a two-step process denoted as DNA leading to transcription of RNA and RNA leading to translation of the protein. Not all genes encode for proteins. Hence, not all DNA gets transcribed to RNA.

A gist of DNA replication:

Initiation of replication starts at a point where the unwinding of DNA takes place. Enzymes synthesize an RNA primer and the fragments so that new nucleotides get added. The segments get elongated through a process of elongation followed by removal of the primers. The unjoined fragments get ligated to end the process of replication. Using DNA as a template, the transcription starts.

RNA synthesis:

Genes are known as ordered sequences of nucleotide bases encoding polypeptide chains via RNA molecules. They are nothing but the pieces of DNA that consist of specific information for making a particular protein. Each gene associates with regulatory sequences known as gene regulatory elements. They are involved in regulation of transcription. The process of transcription starts with denaturation of the DNA double helix. The enzyme known as RNA polymerase catalyzes the process of transcription. In prokaryotes, RNA polymerase is responsible for unwinding. The process of unwinding in eukaryotes occurs with the help of other proteins. The DNA starts unwinding next to the gene involved the process. Hence, RNA polymerase starts catalyzing the RNA synthesis. The RNA gets synthesized from 5’-3’ direction along the 3’-5’ template strand. Out of the two DNA strands, only one strand participates in the process of transcription. Total four nucleotide phosphates act as precursors for transcription. They include ATP, GTP, CTP, and UTP. Recall that DNA synthesis requires RNA primers. However, RNA synthesis does not require primers. RNA polymerases are efficient in initiating the synthesis of new polynucleotide chains without any primers. Participant molecules in RNA synthesis are DNA strand, RNA polymerases, nucleoside triphosphates, elongation and termination factors. Also, there is a requirement of specific gene sequences for the initiation of transcription. In both prokaryotes and eukaryotes, the process of transcription occurs in three consecutive steps such as initiation, elongation, and termination. The eukaryotic transcription needs an understanding of prokaryotic transcription first. 
A prokaryotic gene responsible for transcription needs the three following sequences:

·    The promoter sequence is an upstream sequence present at the start of the RNA coding gene sequences.

·   RNA coding gene sequences are known as DNA sequences capable of getting transcribed to RNA.

·        The terminator specifies the destination point of transcription.

The genes specifying the initiation process have two promoter sequences known as -35 and -10. Each promoter has a specific consensus sequence.

Promoter region	No. of base pairs upstream	Consensus sequence
-35	35	5’-TTGACA-3’
-10	10	5’-TATAAT-3’

Table: Promoters -35 and -10 respectively.

Initiation:

RNA synthesis gets initiated by the recruitment of RNA polymerase holoenzyme. It binds to the promoter region. The holoenzyme consists of the core enzyme form of RNA polymerase. It has four polypeptides such as two α, one β, and one β’ polypeptides bound to a sigma factor. It recognizes both the promoter regions. It first recognizes -35 region where the DNA is a close promoter complex. Then the holoenzyme unwinds the DNA. The untwisted form of the promoter is known as an open promoter complex. The RNA polymerase orients itself to the Pribnow box for further process. Thus, the process of initiation includes two main steps. First, RNA polymerase gets attached to its core promoter. Second, the closed promoter gets converted into an open promoter complex. Then the RNA gets synthesized. For a successful initiation, RNA polymerase now moves away from the promoter region.

Image: Transcription in prokaryotes

Elongation and termination

 The RNA polymerase approximately includes a 30 base pair of DNA. It includes a transcription bubble of 12-14 base pairs. The RNA gets attached to the template strand of the DNA. RNA-DNA base pairs assist the RNA in the attachment. The main feature of RNA polymerase observed during the elongation process does not constantly synthesize the transcript. Instead, it follows a discontinuous synthesis. The process of elongation although rapid gets interspersed by brief pauses. During brief pauses, the active site of the polymerase undergoes a slight structural conformation or gets rearranged. A pause lasts for a few milliseconds and accompanies backtracking thereby occurring randomly. A pause helps in termination of the transcript synthesis. The termination of transcription follows a signal given by terminator sequences. An important protein involved in the process of termination is known as rho (ρ). Hence, there are two types of terminator sequences known as Rho-dependent and Rho-independent terminators. The Rho-dependent terminators are known as type II terminators. They lack A-T string and avoid forming hairpin loops. Rho has RNA binding ATPase domain. Rho-independent terminators, on the other hand, form hairpin loops and consist of inverted repeat sequences. A string of AT base pairs transcribes a string of Us. When the inverted palindrome (with a hairpin loop) gets transcribed, there arises RNA-RNA base pairing. Studies revealed the presence of a flap structure on the surface of RNA polymerase which mediates in termination.

Anti-termination:

It is a process beyond termination. There is an anti-termination process too common in prokaryotes. Anti-termination occurs when RNA polymerase ignores a terminator signal and continues to elongate the transcript until the next signal. Anti-termination provides a mechanism through which few genes at the end of the operon get switched off. Anti-termination protein attaches to the DNA and transfers to the RNA polymerase. However, the reason behind switching off those genes remains unclear.

Premature termination and attenuation:

The primary transcript produced by RNA polymerase is known as a mature mRNA. The bacteria involve coupled transcription and translation. It allows a special type of control known as attenuation. It is a process in which the expression of amino acid biosynthesis gets regulated. Some of the bacteria get bacteriophage infection. Bacteriophages transcribe their genomes using bacterial RNA polymerases.

References:

[1] Transcription, William M. Brown, Philip M. Brown
[2] Molecular Biology of the Cell, Bruce Alberts

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DNA Replication in Prokaryotes

Replication is a process of DNA synthesis or DNA copying. It follows the semiconservative type of replication. A semiconservative replication results into two double-stranded DNA molecules. Each DNA has one strand retained from the parent and one newly synthesized strand. Various experiments tried revealing the mechanisms of replication. The process of replication in prokaryotes is clear in E. coli bacteria.

The process of DNA replication involves three steps such as initiation, elongation, and termination. The DNA replication requires replication enzymes and certain proteins. Arthur Kornberg and his colleagues worked on bacterial DNA replication and identified the DNA replication enzymes. They studied replication in E. coli.

Initiation of replication:

A DNA sequence known as the replicator directs the initiation of replication. This replicator includes the origin of replication. The origin of replication is known as Ori C. It is a specific region where the DNA denaturation occurs. Thus, it is a point at which the loss of native conformation of DNA results into the single-stranded structure, making the process of replication easier. The local DNA denaturation results in DNA replication bubble, leading to segments of single strands known as the template strands. The Y-shaped replication fork arises due to the exposure of two template strands. At this point, the two DNA strands get separated. A sequentially replicating segment of DNA is known as a replicon. The bacterial chromosomes contain a single replicator. The replication fork moves in the direction of untwisting of the DNA. There arise two replication forks while untwisting. Hence, the process of DNA replication is bidirectional. The Ori C is an AT-rich region. It gets denatured very easily.

An initiator DNA-a protein binds to the replicator. Thus, the DNA replication gets initiated due to stimulation of the denaturation process. The DNA helicase starts untwisting the DNA bi-directionally. It leads to the release of energy by hydrolysis of ATP. The energy gets utilized for untwisting. Then comes the role of DNA primases. The enzyme DNA primase forms a primosome complex. This enzyme synthesizes a short RNA primer. New nucleotides get added to the primer. At each replication fork, 10 to 100 nucleotides or pairs get added per second. The single strands created by the unwinding of DNA get stabilized by single-strand binding protein (SSB). Since there are two strands, one at the top and one at the bottom, the synthesis of RNA primer occurs on both the templates. The RNA primers get further lengthened by DNA polymerase III.

Okazaki fragments:

The DNA strands get synthesized from 5’ to 3’ direction at the Y-shaped replication fork. Two types of strands get synthesized. Leading strand is known as a continuous strand. A discontinuous strand is known as a lagging strand. The lagging strand gets synthesized in the form of discontinuous fragments known as Okazaki fragments.

Since the leading strand gets synthesized continuously, and the lagging strand is discontinuous, the process of replication is known as a semi-discontinuous type of replication. The two Okazaki strands get joined by DNA ligase enzyme. 

Image: DNA Replication steps: The image describes five main steps of DNA replication in prokaryotes. (1)The unwinding of the DNA occurs due to helicase activity. The replication fork proceeds further. Two types of strands form including the leading strand and the lagging strand. The SSBPs, primase, and the DNA polymerase-III get recruited. These components allow new DNA synthesis. (2) In this step, the DNA polymerase-III get dissociated. Discontinuous strand synthesis starts on the lagging strands. These strands occur in the form of fragments. Thus, they are known as Okazaki fragments. (3) The replication fork gets extended little more. Thus, the discontinuous strand synthesis proceeds further. (4) The DNA polymerase-I replaces the RNA primer. (5) DNA ligase seals the gaps.

The gist of events occurring at the replication fork:

1.     DNA primase synthesizes an RNA primer.

2.     Replication of the lagging strand occurs.

3.     Untwisting and elongation of new DNA strands.

4.     Primer removal by DNA polymerase I.

5.     Ligation of the adjacent fragments.

Circular DNA replicates bidirectionally:

The E. coli parental strand remains circular. These circular forms exhibit theta shaped structures. These structures arise as replication bubbles. As the two DNA strands untwist, the positive supercoils arise. Hence, the rotation helps in moving the molecule ahead. Topoisomerase solves the problem of supercoiling by introducing the negative supercoils. It keeps both the parent strands intact during replication. Hence, the unreplicated part undergoes a repeated negative supercoiling.

Rolling circle model:

It efficiently synthesizes multiple circular genome copies. Lambda bacteriophage uses this type of replication. A nick gets initiated at one of the parent strands. Then the strand rolls of a linear copy of the circular genome. The synthesis of the second strand converts the linear single-stranded genome into a double-stranded DNA.

References:

[1] IGenetics, Peter Russell, second edition

[2] DNA Replication, Arthur Kornberg, ‎Tania A. Baker, 2005
[3] The Cell Cycle: Principles of Control, Page 61, David Morgan, ‎David Owen Morgan, 2007

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