Transcription and translation in prokaryotes and eukaryotes pdf

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Transcription and translation in prokaryotes and eukaryotes pdf

Gusarov, I., & Nudler, E. The mechanism of intrinsic transcription termination. Molecular Cell 3, 495?504 (1999) Mandal, M., & Breaker, R. R. Gene regulation by riboswitches. Nature Reviews Molecular Cell Biology 5, 451?463 (2004) (link to article) Merino, E., & Yanofsky, C. Transcription attenuation: A highly conserved regulatory strategy used by bacteria. Trends in Genetics 21, 260?264 (2005) Yarnell, W. S., & Roberts, J. W. Mechanism of intrinsic transcription termination and anti-termination. Science 284, 611?615 (1999) Yanofsky, C. Attenuation in the control of expression of bacterial operons. Nature 289, 751?758 (1981) doi:10.1038/289751a0 (link to article) Page ID9282 Contributed by BoundlessGeneral Microbiology at Boundless Prokaryotic transcription is the process in which messenger RNA transcripts of genetic material in prokaryotes are produced, to be translated for the production of proteins. Prokaryotic transcription occurs in the cytoplasm alongside translation. Prokaryotic transcription and translation can occur simultaneously. This is impossible in eukaryotes, where transcription occurs in a membrane-bound nucleus while translation occurs outside the nucleus in the cytoplasm. In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm. Figure: Protein synthesis: An overview of protein synthesis.Within the nucleus of the cell (light blue), genes (DNA, dark blue) are transcribed into RNA. This RNA is then subject to post-transcriptional modification and control, resulting in a mature mRNA (red) that is then transported out of the nucleus and into the cytoplasm (peach), where it undergoes translation into a protein. mRNA is translated by ribosomes (purple) that match the three-base codons of the mRNA to the three-base anti-codons of the appropriate tRNA. Newly synthesized proteins (black) are often further modified, such as by binding to an effector molecule (orange), to become fully active. Transcription is controlled by a variety of regulators in prokaryotes. Many of these transcription factors are homodimers containing helix-turn-helix DNA-binding motifs. The following steps occur, in order, for transcription initiation: RNA polymerase (RNAP) binds to one of several specificity factors, , to form a holoenzyme. In this form, it can recognize and bind to specific promoter regions in the DNA. The -35 region and the -10 ("Pribnow box") region comprise the basic prokaryotic promoter, and |T| stands for the terminator. The DNA on the template strand between the +1 site and the terminator is transcribed into RNA, which is then translated into protein. At this stage, the DNA is double-stranded ("closed"). This holoenzyme/wound-DNA structure is referred to as the closed complex. The DNA is unwound and becomes single-stranded ("open") in the vicinity of the initiation site (defined as +1). This holoenzyme/unwound-DNA structure is called the open complex. The RNA polymerase transcribes the DNA (the beta subunit initiates the synthesis), but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the -factor.The -factor eventually dissociates from the holoenzyme, and elongation proceeds. Promoters can differ in "strength"; that is, how actively they promote transcription of their adjacent DNA sequence. Promoter strength is in many (but not all) cases, a matter of how tightly RNA polymerase and its associated accessory proteins bind to their respective DNA sequences. The more similar the sequences are to a consensus sequence, the stronger the binding is. Additional transcription regulation comes from transcription factors that can affect the stability of the holoenzyme structure at initiation. Most transcripts originate using adenosine-5-triphosphate (ATP) and, to a lesser extent, guanosine-5-triphosphate (GTP) (purine nucleoside triphosphates) at the +1 site. Uridine-5-triphosphate (UTP) and cytidine-5-triphosphate (CTP) (pyrimidine nucleoside triphosphates) are disfavoured at the initiation site. Two termination mechanisms are well known: Intrinsic termination (also called Rho-independent transcription termination) involves terminator sequences within the RNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template. Rho-dependent termination uses a termination factor called factor(rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho utilisation site on the nascent RNA strand and runs along the mRNA towards the RNAP. A stem loop structure upstream of the terminator region pauses the RNAP, when -factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription. In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm. Transcription is known to be controlled by a variety of regulators in prokaryotes. Many of these transcription factors are homodimers containing helix-turnhelix DNA -binding motifs. Additional transcription regulation comes from transcription factors that can affect the stability of the holoenzyme structure at initiation. transcription: The synthesis of RNA under the direction of DNA. The prokaryotes, which include bacteria and archaea, are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. A bacterial chromosome is a covalently closed circle that, unlike eukaryotic chromosomes, is not organized around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid. In addition, prokaryotes often have abundant plasmids, which are shorter circular DNA molecules that may only contain one or a few genes. Plasmids can be transferred independently of the bacterial chromosome during cell division and often carry traits such as antibiotic resistance. Because of these unique features, transcription and gene regulation is somewhat different between prokaryotic cells and eukaryotic ones. Learning Objectives Understand the basic steps in the transcription of DNA into RNA in prokaryotic cells Understand the basics of prokaryotic translation and how it differs from eukaryotic translation Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events occurring concurrently on the same DNA template. Prokaryotic transcription often covers more than one gene and produces polycistronic mRNAs that specify more than one protein. Our discussion here will exemplify transcription by describing this process in Escherichia coli, a well-studied bacterial species. Although some differences exist between transcription in E. coli and transcription in archaea, an understanding of E. coli transcription can be applied to virtually all bacterial species. Prokaryotic RNA Polymerase Prokaryotes use the same RNA polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted , , , and comprise the polymerase core enzyme. These subunits assemble every time a gene is transcribed, and they disassemble once transcription is complete. Each subunit has a unique role; the two subunits are necessary to assemble the polymerase on the DNA; the -subunit binds to the ribonucleoside triphosphate that will become part of the nascent "recently born" mRNA molecule; and the binds the DNA template strand. The fifth subunit, , is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Without , the core enzyme would transcribe from random sites and would produce mRNA molecules that specified protein gibberish. The polymerase comprised of all five subunits is called the holoenzyme (a holoenzyme is a biochemically active compound comprised of an enzyme and its coenzyme). Prokaryotic Promoters Figure 1. The subunit of prokaryotic RNA polymerase recognizes consensus sequences found in the promoter region upstream of the transcription start sight. The subunit dissociates from the polymerase after transcription has been initiated. A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription. In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among prokaryotic genomes, a few elements are conserved. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species (Figure 1). The -10 consensus sequence, called the -10 region, is TATAAT. The -35 sequence, TTGACA, is recognized and bound by . Once this interaction is made, the subunits of the core enzyme bind to the site. The A?T-rich -10 region facilitates unwinding of the DNA template, and several phosphodiester bonds are made. The transcription initiation phase ends with the production of abortive transcripts, which are polymers of approximately 10 nucleotides that are made and released. View this MolecularMovies animation to see the first part of transcription and the base sequence repetition of the TATA box. Elongation and Termination in Prokaryotes The transcription elongation phase begins with the release of the subunit from the polymerase. The dissociation of allows the core enzyme to proceed along the DNA template, synthesizing mRNA in the 5 to 3 direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it (Figure 2). The base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely. Figure 2. Click for a larger image. During elongation, the prokaryotic RNA polymerase tracks along the DNA template, synthesizes mRNA in the 5 to 3 direction, and unwinds and rewinds the DNA as it is read. Prokaryotic Termination Signals Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals. One is protein-based and the other is RNA-based. Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble. Rho-independent termination is controlled by specific sequences in the DNA template strand. As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C?G nucleotides. The mRNA folds back on itself, and the complementary C?G nucleotides bind together. The result is a stable hairpin that causes the polymerase to stall as soon as it begins to transcribe a region rich in A?T nucleotides. The complementary U?A region of the mRNA transcript forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript. Upon termination, the process of transcription is complete. By the time termination occurs, the prokaryotic transcript would already have been used to begin synthesis of numerous copies of the encoded protein because these processes can occur concurrently. The unification of transcription, translation, and even mRNA degradation is possible because all of these processes occur in the same 5 to 3 direction, and because there is no membranous compartmentalization in the prokaryotic cell (Figure 3). In contrast, the presence of a nucleus in eukaryotic cells precludes simultaneous transcription and translation. Figure 3. Multiple polymerases can transcribe a single bacterial gene while numerous ribosomes concurrently translate the mRNA transcripts into polypeptides. In this way, a specific protein can rapidly reach a high concentration in the bacterial cell. Visit this BioStudio animation to see the process of prokaryotic transcription. Which subunit of the E. coli polymerase confers specificity to transcription? The -10 and -35 regions of prokaryotic promoters are called consensus sequences because ________. they are identical in all bacterial species they are similar in all bacterial species they exist in all organisms they have the same function in all organisms Prokaryotic Translation Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between bacterial and eukaryotic translation. Initiation The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-tRNAfMet) (Figure 4). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome. In eukaryotes, initiation complex formation is similar, with the following differences: The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5 cap of the eukaryotic mRNA, then tracks along the mRNA in the 5 to 3 direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit. Figure 4. Translation in bacteria begins with the formation of the initiation complex, which includes the small ribosomal subunit, the mRNA, the initiator tRNA carrying N-formyl-methionine, and initiation factors. Then the 50S subunit binds, forming an intact ribosome. Elongation In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet-tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG. Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3 direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds. Termination The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation init iation complex. In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure 5 and listed in Table 1. Figure 5. (a) In prokaryotes, the processes of transcription and translation occur simultaneously in the cytoplasm, allowing for a rapid cellular response to an environmental cue. (b) In eukaryotes, transcription is localized to the nucleus and translation is localized to the cytoplasm, separating these processes and necessitating RNA processing for stability. Table 1. Comparison of Translation in Bacteria Versus Eukaryotes Property Bacteria Eukaryotes Ribosomes 70S 30S (small subunit) with 16S rNA subunit 50S (large subunit) with 5S and 23S rRNA subunits 80S 40S (small subunit) with 18S rRNA subunit 60S (large subunit) with 5S, 5.8S, and 28S rRNA subunits Amino acid carried by initiator tRNA fMet Met Shine-Dalgarno sequence in mRNA Present Absent Simultaneous transcription and translation Yes No Check Your Understanding Answer the question(s) below to see how well you understand the topics covered in the previous section. This short quiz does not count toward your grade in the class, and you can retake it an unlimited number of times. Use this quiz to check your understanding and decide whether to (1) study the previous section further or (2) move on to the next section.

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