Transcription and Translation in Prokaryotes

Stages of transcription in prokaryotes

As with eukaryotes, prokaryotic transcription takes place in three steps: initiation, elongation, and termination. One of the major differences between eukaryotic and prokaryotic transcription is that in eukaryotes, this process happens in the nucleus, while prokaryotes transcribe their genes in the cytoplasm (they do not have a nucleus).

Other important differences between prokaryotic and eukaryotic transcription can be found in the initiation and termination stages. We'll go through each stage and identify where these differences lie.

Transcription initiation in prokaryotes

Initiation of transcription begins with the enzyme RNA polymerase binding to a specific sequence on the DNA double-strand known as the promoter, which represents the start of the gene. Unlike eukaryotes which have three types of polymerase (I, II, and III), prokaryotes have only one type of polymerase.

The DNA then unwinds at the promoter region, and the RNA polymerase binds to the transcription start site. Now, the RNA polymerase is ready to “read” the bases in the sequence of the unwound DNA strand and produce RNA with a complementary base sequence.

Prokaryotic cells have operators, repressors, and activator proteins that participate in the initiation stage.

• Operators are sequences that instruct proteins called repressors to bind to the DNA ahead of the transcription start site.

• Repressors prevent the RNA polymerase from accessing the DNA. Because the RNA polymerase is physically blocked, transcription cannot take place.

• Activator proteins send signals to the cell if and when gene expression is needed. When they do, repressors are removed from obstructing the RNA polymerase.

Transcription termination in prokaryotes

The RNA polymerase “reads” the bases by travelling through the DNA strand from the 3′ end to the 5’ end (\(3' \rightarrow 5'\)). As it travels through the strand, it “copies” the strand by adding complementary base pairs from the opposite direction.

Because the end-product of transcription is RNA, the base adenine (A) is encoded as (complemented by) uracil (U) instead of thymine (T) when the complementary bases are added.

It is also in this stage that the sugar-phosphate backbone of the RNA is created by the RNA polymerase. Whereas the pentose sugar in DNA is deoxyribose, the resulting RNA strand will have ribose.

Transcription termination in prokaryotes

Elongation continues until the RNA polymerase encounters a termination sequence in the gene which signals the end of transcription. Prokaryotic termination can follow two possible paths:

• Rho-independent termination: when the RNA polymerase crosses a palindromic termination sequence that produces a stem-loop structure, the weak connection between the RNA-DNA hybrid and the polymerase causes it to dissociate.

• Rho-dependent termination: A protein called rho binds to the transcribed mRNA and travels along the strand toward the polymerase. When it reaches the polymerase, it induces the mRNA to dissociate from the polymerase.

In this stage, the hydrogen bonds that bring together the RNA and DNA helices break, releasing the newly formed RNA. In prokaryotic cells, the transcription process ends here, but in eukaryotic cells, the mRNA undergoes further processing.

Stages of translation in prokaryotes

As with eukaryotes, prokaryotic translation takes place in three steps: initiation, elongation, and termination. In both eukaryotes and prokaryotes, translation takes place in the cytoplasm with the mediation of the ribosomes. The major difference between prokaryotic and eukaryotic translation is in the initiation stage, while the elongation and termination stages are very similar.

Again, we'll discuss the entire prokaryotic translation process and identify where these differences lie.

Translation initiation in prokaryotes

Ribosomes can be broken down into large and small ribosomal subunits.

• The small ribosomal subunit binds the mRNA template.

• The large ribosomal subunit binds the transfer RNAs (tRNAs).

In prokaryotes, the small ribosomal subunit binds to the Shine-Dalgarno sequence on the mRNA template. The Shine-Dalgarno sequence “AGGAGG” is found just ahead of the “AUG” start codon.

The small ribosomal subunit then binds to the charged initiator tRNA molecule (tRNAi) and together these traverse the mRNA strand up to the start codon. This signals the start of translation.

The anticodon on the tRNAi binds to the start codon through base pairing. Then, the small ribosomal subunit, mRNA, and tRNAi attach to the large ribosomal subunit, forming what is known as the initiation complex.

Translation elongation in prokaryotes

The basics of the elongation and termination stages of prokaryotic and eukaryotic translation are similar.

During elongation, the ribosome continues to translate codons and add amino acids to the growing amino acid chain. Elongation takes place in the three compartments of the large ribosomal subunit: A (aminoacyl) site, P (peptidyl) site, and E (exit) site.

The process of elongation can be summarized as follows:

• Methionine-carrying tRNAi binds to the P site while aminoacyl-carrying tRNA binds to the A site.

• The energy-carrying molecule guanosine triphosphate (GTP), which is bound to the elongation factor, is hydrolyzed, releasing the elongation factor from the ribosome.

• A peptide bond forms between the methionine-carrying tRNAi and the aminoacyl-carrying tRNA.

• Methionine travels to the A site and forms a peptidyl tRNA.

• The dissociated tRNAi at the P site is moved to the E site.

• The ribosome moves the next codon in the free A site.

• As the ribosome travels along the mRNA strand, it continues to register each codon, adding the corresponding charged tRNA anticodon to the chain.

• Elongation continues until the entire mRNA is translated into a polypeptide chain.

Translation termination in prokaryotes

Translation ends when a nonsense or stop codon (UAA, UAG, or UGA) enters the A site. Release factors call for the tRNA and the polypeptide chain to be hydrolyzed, releasing the newly formed polypeptide chain. During and after translation, the polypeptide chain is “folded” into its specific three-dimensional structure in a process called protein folding.

After termination, the nucleotides of the degraded mRNA can participate in another transcription reaction while the small and large ribosomal subunits dissociate from each other and from the mRNA, allowing them to participate in another translation process.

Coupled transcription and translation in prokaryotes

Instead of being enclosed in the nucleus, prokaryotic DNA is suspended in the cytoplasm in the central region of the cell called nucleoid. Because there is no membrane that separates prokaryotic DNA from ribosomes, transcription and translation can take place almost simultaneously in prokaryotes (Fig. 1). Specifically, ribosomes can begin translation even when the transcription of mRNA has not yet ended, forming RNA polymerase-mRNA-ribosome complexes.

On the other hand, eukaryotic DNA is enclosed in the membrane-bound nucleus, separating it from the ribosomes in the cytoplasm and endoplasmic reticulum. Because of this eukaryotic transcription and translation take place separately: the transcription of DNA into mRNA takes place inside the nucleus. From the nucleus, the mRNA moves to the ribosomes in the cytoplasm and endoplasmic reticulum where it is translated into protein (Fig. 2).

Transcription factors in prokaryotes

Transcription factors are proteins that regulate the activity of a gene by sending a signal to the cell that transcription is needed. As such, they play an important role in regulating prokaryotic gene expression.

Prokaryotes such as bacteria use a transcription factor called sigma which loosely binds the DNA and assists the RNA polymerase in searching for a promoter during the initiation stage.

The sigma factor dissociates from the RNA polymerase once the transcription has initiated, allowing the RNA polymerase to continue the elongation process. Thus, the sigma factor plays a critical role in regulating prokaryotic gene expression by controlling the specificity of RNA polymerase binding to the promoter regions.

There are multiple types of sigma factors in prokaryotic cells, each recognizing a different set of promoter sequences and regulating the transcription of specific genes. For example, the sigma-70 factor is the most common sigma factor in Escherichia coli, and it recognizes promoters for housekeeping genes involved in essential cellular functions. In contrast, alternative sigma factors, such as sigma-32, are induced under stress conditions and regulate the transcription of stress response genes.

Differences between transcription and translation in prokaryotes and eukaryotes

Like eukaryotic cells, prokaryotic cells undergo gene expression and regulation. Prokaryotic cells are simple, unicellular organisms that do not have a nucleus or any other membrane-bound organelles.

This distinguishing feature of prokaryotic cells makes the transcription and translation process of prokaryotic cells different from those of eukaryotic cells. We have already covered most of the differences, but there is another important process that differs between prokaryotes and eukaryotes: DNA repair, specifically transcription-coupled repair (TCR).

TCR is a DNA repair mechanism by which the RNA polymerase detects lesions in the DNA that is being transcribed and stops the transcription. This avoids creating faulty messenger RNA and proteins. Additionally, the RNA polymerase recruits repair proteins that will attempt to fix the error in the DNA, so that mutations are not permanently embedded in the DNA.