Prokaryotic Transcription- Enzymes, Steps, Significance

DNA and RNA are two types of nucleic acids that store and transmit genetic information in living cells. They are composed of linear chains of nucleotides, which are the building blocks of nucleic acids. Each nucleotide consists of three components: a nitrogenous base, a pentose sugar, and a phosphate group.

DNA stands for deoxyribonucleic acid. It is the main genetic material in most organisms, except for some viruses that use RNA as their genome. DNA has a double-helical structure, with two strands of nucleotides coiled around each other and held together by hydrogen bonds between complementary bases. The four bases in DNA are adenine (A), thymine (T), guanine (G), and cytosine (C). A pairs with T, and G pairs with C. The sugar in DNA is deoxyribose, which lacks an oxygen atom at the 2` position of the ring.

RNA stands for ribonucleic acid. It is a single-stranded molecule that can fold into various shapes and forms. RNA has many functions in the cell, such as carrying genetic information from DNA to the ribosomes for protein synthesis (messenger RNA or mRNA), helping in the assembly and function of ribosomes (ribosomal RNA or rRNA), transporting amino acids to the ribosomes for protein synthesis (transfer RNA or tRNA), regulating gene expression (small interfering RNA or siRNA, microRNA or miRNA, etc.), and catalyzing biochemical reactions (ribozymes). The four bases in RNA are adenine (A), uracil (U), guanine (G), and cytosine (C). A pairs with U, and G pairs with C. The sugar in RNA is ribose, which has an oxygen atom at the 2` position of the ring.

The main enzyme responsible for transcription in prokaryotes is RNA polymerase, which synthesizes RNA from a DNA template. RNA polymerase is a large and complex enzyme that consists of several subunits with different functions. The subunits are:

• Two α subunits, which help to assemble the enzyme and bind to regulatory factors.
• One β subunit, which contains the active site for catalyzing the formation of phosphodiester bonds between ribonucleotides.
• One β` subunit, which binds to the DNA template and unwinds it during transcription.
• One ω subunit, which stabilizes the enzyme and protects it from degradation.
• One σ subunit, which recognizes specific promoter sequences on the DNA and initiates transcription.

The complete enzyme with all six subunits is called the holoenzyme, while the enzyme without the σ subunit is called the core enzyme. The σ subunit can dissociate from the holoenzyme after transcription initiation and reassociate with another core enzyme to start a new round of transcription. There are different types of σ subunits that can bind to different promoters and regulate gene expression in response to environmental signals.

RNA polymerase does not need a primer to start transcription, unlike DNA polymerase. It can initiate transcription at any point on the DNA template that has a promoter sequence. It then adds ribonucleotides to the 3` end of the growing RNA chain, using the complementary base pairing rules (A with U and C with G). The RNA polymerase moves along the DNA template in the 3` to 5` direction, synthesizing RNA in the 5` to 3` direction. The RNA transcript has the same sequence as the non-template (or coding) strand of DNA, except that it has U instead of T.

RNA polymerase can also terminate transcription when it reaches a specific signal on the DNA template. There are two main types of termination signals in prokaryotes: intrinsic and rho-dependent. Intrinsic termination signals consist of a GC-rich palindrome followed by an AT-rich sequence on the DNA template. The RNA transcript forms a hairpin loop at the palindrome and then dissociates from the DNA template at the weak AU bonds. Rho-dependent termination signals require an additional protein called rho, which binds to a specific sequence on the RNA transcript and moves along it until it reaches the RNA polymerase. Rho then interacts with the RNA polymerase and causes it to release the RNA transcript and detach from the DNA template.

RNA polymerase is not the only enzyme involved in transcription in prokaryotes. There are also other enzymes that modify or process the RNA transcripts after they are synthesized. For example, some enzymes remove extra nucleotides from the ends of tRNA and rRNA molecules, while others add chemical groups such as methyl or pseudouridine to enhance their stability and function. Some enzymes also splice out introns from mRNA molecules, although this is rare in prokaryotes compared to eukaryotes.

The initiation phase of transcription is the first step in which RNA polymerase recognizes and binds to a specific site on the DNA, called a promoter site. The promoter site is located upstream from the gene that will be transcribed and determines which strand of DNA will be used as the template and where transcription will start.

The promoter site consists of two conserved sequences that are important for RNA polymerase recognition and binding: the -10 sequence (also known as the Pribnow box) and the -35 sequence. These sequences are named according to their positions relative to the transcription start site (+1). The -10 sequence has the consensus TATAAT and the -35 sequence has the consensus TTGACA. The distance between these two sequences is usually 17-19 bp.

The RNA polymerase holoenzyme, which consists of five subunits (α2ββ`ω) and a σ factor (α2ββ`ωσ), binds to the promoter site with the help of the σ factor. The σ factor is responsible for recognizing and interacting with the -10 and -35 sequences and conferring specificity to RNA polymerase. Different σ factors can recognize different promoters and regulate different sets of genes.

Once the RNA polymerase holoenzyme binds to the promoter site, it causes local unwinding of the DNA double helix, forming a transcription bubble of about 17 bp. This exposes the template strand of DNA for RNA synthesis. The RNA polymerase does not need a primer to initiate transcription; it can start directly from the +1 nucleotide, which is usually a purine (A or G).

The initiation phase ends when the RNA polymerase clears the promoter site and enters the elongation phase. During this transition, the σ factor dissociates from the RNA polymerase core enzyme (α2ββ`ω), which continues to synthesize RNA along the template strand. The σ factor can then bind to another RNA polymerase holoenzyme and initiate transcription at another promoter site.

Promoters are specific DNA sequences that act as binding sites for RNA polymerase and determine where and when transcription will start. Promoters are located upstream of the gene that will be transcribed, usually within 40-60 bp from the transcription start site (TSS). Promoters have two conserved elements: the -10 element and the -35 element, which are recognized by the sigma (σ) subunit of RNA polymerase.

The -10 element, also known as the Pribnow box, has the consensus sequence TATAAT and is located about 10 bp upstream of the TSS. The -35 element has the consensus sequence TTGACA and is located about 35 bp upstream of the TSS. These elements are important for DNA unwinding and RNA polymerase binding during transcription initiation.

The initiation of transcription involves the following steps:

1. The holoenzyme, which consists of the core enzyme (α2ββ`ω) and the σ subunit (α2ββ`ωσ), binds to the promoter region and forms a closed complex with the DNA.
2. The holoenzyme unwinds a short segment of DNA around the -10 element and forms an open complex with the DNA. This exposes the template strand for RNA synthesis.
3. The holoenzyme catalyzes the addition of the first nucleotide (usually a purine) to the 3` end of a growing RNA chain. No primer is required for this step.
4. The holoenzyme extends the RNA chain by adding more nucleotides to the 3` end, using the template strand as a guide. The σ subunit dissociates from the holoenzyme after about 8-10 nucleotides have been added, leaving behind the core enzyme to continue elongation.

The initiation phase is complete when a short RNA transcript has been synthesized and the core enzyme has escaped from the promoter region. The rate of initiation is influenced by several factors, such as the strength of the promoter, the availability of σ factors, and the presence of regulatory proteins.

After transcription initiation, the σ factor is released from the transcriptional complex to leave the core enzyme (α2 ββω) which continues elongation of the RNA transcript. The core enzyme contains the catalytic site for polymerization, probably within the β subunit. The first nucleotide in the RNA transcript is usually pppG or pppA. The RNA polymerase then synthesizes RNA in the 5’ →3’ direction, using the four ribonucleoside 5-triphosphates (ATP, CTP, GTP, UTP) as precursors. The 3-OH at the end of the growing RNA chain attacks the α phosphate group of the incoming ribonucleoside 5-triphosphate to form a 3’5′ phosphodiester bond.

The complex of RNA polymerase, DNA template and new RNA transcript is called a ternary complex (i.e. three components) and the region of unwound DNA that is undergoing transcription is called the transcription bubble. The RNA transcript forms a transient RNA–DNA hybrid helix with its template strand but then peels away from the DNA as transcription proceeds. The DNA is unwound ahead of the transcription bubble and after the transcription complex has passed, the DNA rewinds.

Thus, during the elongation, the RNA polymerase uses the antisense (-) strand of DNA as template and synthesizes a complementary RNA molecule. The RNA produced has the same sequence as the non-template strand, called the sense (+) strand (or coding strand) except that the RNA contains U instead of T. At different locations on the bacterial chromosome, sometimes one strand is used as template, sometimes the other, depending on which strand is the coding strand for the gene in question. The correct strand to be used as template is identified for the RNA polymerase by the presence of the promoter site.

The elongation phase is crucial for ensuring the accuracy and fidelity of transcription. The RNA polymerase has a proofreading mechanism that can correct errors during elongation by removing mismatched nucleotides and resuming synthesis. The rate of elongation varies depending on factors such as temperature, salt concentration and supercoiling of DNA. In E. coli, it has been estimated that the average rate of elongation is about 40 nucleotides per second.

The elongation phase ends when the RNA polymerase encounters a termination signal and ceases transcription, releasing the RNA transcript and dissociating from the DNA. There are two types of termination signals in prokaryotes: intrinsic and rho-dependent. We will discuss them in detail in point 6.

Transcription continues until a termination sequence is reached. The most common termination signal is a GC-rich region that is a palindrome, followed by an AT-rich sequence. The RNA made from the DNA palindrome is self- complementary and so base pairs internally to form a hairpin structure rich in GC base pairs followed by four or more U residues. This hairpin structure causes the RNA polymerase to pause and destabilize the RNA-DNA hybrid, leading to the release of the RNA transcript and the dissociation of the transcription complex.

However, not all termination sites have this hairpin structure. Those that lack such a structure require an additional protein, called rho, to help recognize the termination site and stop transcription. Rho is a hexameric protein that binds to a specific sequence on the RNA transcript called the rho utilization site (rut). Rho then moves along the RNA in the 5` to 3` direction using its ATPase activity until it reaches the RNA polymerase that is stalled at the termination site. Rho then interacts with the RNA polymerase and causes it to release the RNA transcript and dissociate from the DNA template.

Rho-dependent termination is more common in genes that encode proteins involved in energy metabolism, such as glycolysis and respiration. This may be a way for bacteria to regulate their energy production in response to environmental conditions.

In prokaryotes, RNA transcribed from protein-coding genes (messenger RNA, mRNA), requires little or no modification prior to translation. Many mRNA molecules begin to be translated even before RNA synthesis has finished. However, since ribosomal RNA (rRNA) and transfer RNA (tRNA) are synthesized as precursor molecules, they require post-transcriptional processing.

The processing of rRNA and tRNA in prokaryotes involves the following steps:

1. Cleavage of the primary transcript by ribonucleases to generate individual rRNA and tRNA molecules.
2. Modification of some nucleotides by methylation, pseudouridylation, or other chemical reactions.
3. Addition of CCA (cytidine-cytidine-adenosine) sequence at the 3` end of tRNA by a specific enzyme.

The processing of rRNA and tRNA is essential for their proper folding, stability, and function in protein synthesis. The processing enzymes and factors are often specific for different rRNA and tRNA species and may vary among different prokaryotic groups.

The processing of rRNA and tRNA in prokaryotes is different from that in eukaryotes in several aspects:

1. Prokaryotic rRNA and tRNA are transcribed from operons that contain multiple copies of the same or different genes. Eukaryotic rRNA and tRNA are transcribed from single-copy genes or tandem repeats.
2. Prokaryotic rRNA and tRNA are processed in the cytoplasm where they are synthesized. Eukaryotic rRNA and tRNA are processed in the nucleus or nucleolus where they are transcribed and then transported to the cytoplasm.
3. Prokaryotic rRNA and tRNA have fewer and simpler modifications than eukaryotic rRNA and tRNA. Eukaryotic rRNA and tRNA have more diverse and complex modifications that involve more enzymes and factors.

The processing of rRNA and tRNA in prokaryotes is a crucial step for the regulation of gene expression and the adaptation to environmental changes. The processing enzymes and factors can be modulated by various signals such as temperature, nutrient availability, stress, or infection. The processing can also affect the stability, degradation, and interaction of rRNA and tRNA with other molecules.

Transcription of DNA is the method for regulating gene expression in prokaryotes. It occurs in preparation for and is necessary for protein translation. Prokaryotic transcription often covers more than one gene and produces polycistronic mRNAs that specify more than one protein. This allows for the coordinated expression of genes that are involved in the same metabolic pathway or cellular function.

Transcription in prokaryotes is also regulated by various factors, such as the availability of nutrients, environmental signals, and feedback mechanisms. Some genes are constitutively expressed, meaning that they are transcribed at a constant rate regardless of the conditions. Other genes are inducible or repressible, meaning that their transcription can be turned on or off by specific molecules that bind to regulatory proteins or DNA sequences.

Transcription in prokaryotes is also influenced by the structure and topology of the DNA molecule. The DNA in prokaryotes is supercoiled, meaning that it is twisted and compacted to fit inside the cell. Supercoiling affects the accessibility of the DNA to the RNA polymerase and other transcription factors. Some regions of the DNA are more relaxed and easier to transcribe, while others are more tightly wound and harder to transcribe. Supercoiling can also change in response to environmental stress or DNA damage, altering the expression of certain genes.

Transcription in prokaryotes is a fast and efficient process that allows them to adapt to changing conditions and synthesize the proteins they need for survival and growth. By understanding how transcription works in prokaryotes, we can gain insights into their molecular biology, physiology, and evolution. We can also use this knowledge to manipulate their gene expression for biotechnological applications, such as producing recombinant proteins, antibiotics, or biofuels.

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