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A few years after he and James Watson had proposed the double helical structure for DNA, Francis Crick (with other collaborators) proposed that a less stable nucleic acid, RNA, served as a messenger RNA that provided a transient copy of the genetic material that could be translated into the protein product encoded by the gene. Such mRNAs were indeed found. These and other studies led Francis Crick to formulate this “central dogma” of molecular biology (Figure 1).
This model states thatDNA serves as the repository of genetic information. It can bereplicatedaccurately and indefinitely. Thegenetic information is expressedby the DNA first serving as a template for thesynthesis of (messenger) RNA; this occurs in a process calledtranscription. The mRNA then serves as a template, which is read by ribosomes andtranslatedintoprotein. The protein products can be enzymes that catalyze the many metabolic transformations in the cell, or they can be structural proteins.
Figure 1.The central dogma of molecular biology. (Public Domain; Narayanese).
Although there have been some additional steps added since its formulation, the central dogma has stood the test of time and myriad experiments. It provides a strong unifying theme to molecular genetics and information flow in cell biology and biochemistry.
Although in many cases a gene encodes one polypeptide, other genes encode afunctional RNA. Some genes encodetRNAsandrRNAsneeded for translation, others encode other structural and catalytic RNAs. Genes encode some product that is used in the cell, i.e. that when altered generates an identifiable phenotype. More generally, genes encode RNAs, some of which are functional as transcribed (or with minor alterations via processing) such as tRNAs and rRNAs, and others are messengers that are then translated into proteins. These proteins can provide structural, catalytic and regulatory roles in the cell.
Note thestatic role of DNAin this process. Implicit in this model is the idea that DNA does not provide an active cellular function, but rather it encodes macromolecules that are functional. However, the expression of virtually all genes is highly regulated. The sites on the DNA where this control is exerted are indeed functional entities, such as promoters and enhancers. In this case, the DNA is directly functional (cis‑regulatory sites), but the genes being regulated by these sites still encode some functional product (RNA or protein).
Studies of retroviruses lead Dulbecco to argue that the flow of information is not unidirectional, but in fact RNA can be converted into DNA (some viral RNA genomes are converted into DNA proviruses integrated into the genome). Subsequently Temin and Baltimore discovered the enzyme that can make a DNA copy of RNA, i.e. reverse transcriptase.
Transcription from DNA to RNA
Section summary
Bacteria, archaea, and eukaryotes must all transcribe genes from their genomes. While the cellular location may be different (eukaryotes perform transcription in the nucleus; bacteria and archaea perform transcription in the cytoplasm), the mechanisms by which organisms from each of these clades carry out this process arefundamentallythe same and canbe characterizedby three stages: initiation, elongation, and termination.
A shortoverview of transcription
Transcription isthe process ofcreating an RNA copy of a segment of DNA. Since this is aprocess, we want to apply the Energy Story rubric to develop a functional understanding of transcription. What does the system of molecules look like before the start of the transcription? What does it look like at the end? What transformations of matter and transfers of energy happen during the transcription, and what catalyzes the process? We also want to think about the process from aDesign Challengestandpoint. If the biological task is to create a copy of DNA in the chemical language of RNA, what challenges can we reasonably hypothesize or expect, given our knowledge about other nucleotide polymer processes, mustbe overcome? Is there evidence that Nature solved these problems in different ways? What seem to be the criteria for the success of transcription? You get the idea.
Listing some basic requirements for transcription
Let us first consider the tasks at hand by using some of our foundational knowledge and imagining what might need to happen during transcription if the goal is to make an RNA copy of a piece of one strand of a double-stranded DNA molecule. We'll see that using some basic logic allows us to infer many of the important questions and things that we need to knowin orderto describe the process properly.
Let us imagine that we want to design ananomachine/nanobot that would conduct transcription. We can use someDesign Challengethinking to identify problems and subproblems that need tobe solvedby our little robot.
• Where should the machine start? Along the millions to billions of base pairs, where should the machinebe directed?
• Where should the machine stop?
• If we have start and stop sites, we will need ways of encoding that information so that our machine(s) can read this information—how will thatbe accomplished?
• How many RNA copies of the DNA will we need to make?
• How fast do the RNA copies need tobe made?
• How accurately do the copies need tobe made?
• How much energy will the process take and where is the energy going to come from?
These are only some core questions. One can dig deeper if they wish. However, these are already good enough for us to get a good feel for this process. Notice, too, that many of these questions are remarkably similar to those we inferred might be necessary to understand DNA replication.
The building blocks of transcription
The building blocks of RNA
Recall from our discussion on the structure of nucleotides that the building blocks of RNA are very similar to those in DNA. In RNA, the building blocks comprise nucleotide triphosphates thatare composedof a ribose sugar, a nitrogenous base, and three phosphate groups. The key differences between the building blocks of DNA and those of RNA are thatRNA molecules are composedof nucleotides with ribose sugars (as opposed to deoxyribose sugars) and use uridine, a uracil containing nucleotide (as opposed to thymidine in DNA). Note below that uracil and thymine are structurally very similar—the uracil is just lacking a methyl (CH3) functional group compared to thymine.
Figure 2. The basic chemical components of nucleotides.
Attribution:Marc T. Facciotti (original work)
Transcription initiation
Promoters
Proteins responsible for creating an RNA copy of a specific piece of DNA (transcription) must first be able to recognize the beginning of the element tobe copied. Apromoteris a DNA sequence onto which various proteins, collectively known as the transcription machinery, bind and starttranscription. In most cases, promoters exist upstream (5' to the coding region) of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the cell transcribes the corresponding coding portion of the gene all the time, sometimes, or infrequently. Although promoters vary among species, a few elements of similar sequencesare sometimes conserved. At the-10and-35regions upstream of the initiation site, there are twopromoterconsensussequences, or regions similar across many promoters and across various species. Some promoters will have a sequence very similar to the consensus sequence (the sequence containing the most common sequence elements), and others will look very different. These sequence variations affect the strength to which the transcriptional machinery can bind to the promoter to start transcription. This helps to control the number of transcripts thatare madeand how often they get made.
Figure 3. (a) A general diagram of a gene. The gene includes the promoter sequence, an untranslated region (UTR), and the coding sequence. (b) A list of several strong E. colipromotersequences. The -35 box and -10 boxare highly conservedsequences throughout the strong promoter list. Weaker promoters will have more base pair differences when compared to these sequences.
Source:http://www.discoveryandinnovation.co...lecture12.html
Bacterial vs. eukaryotic promoters
In bacterial cells, the -10 consensus sequence, called the -10 region, is AT rich, often TATAAT. The -35 sequence, TTGACA,is recognizedand bound by the proteinσ. Once this protein-DNA interactionis made, the subunits of the core RNA polymerase bind to the site. Because of the relatively lower stability of AT associations, the AT-rich -10 region facilitates unwinding of the DNA template, andseveralphosphodiesterbonds are made.
Eukaryotic promoters are much larger and more complex than prokaryotic promoters, but both have an AT-rich region—in eukaryotes, itis typically calleda TATA box. For example, in the mouse thymidine kinase gene,the TATA box is locatedatapproximately -30. For this gene, the exact TATA box sequence is TATAAAA, as read in the 5' to 3' direction on thenontemplatestrand. This sequence is not identical to theE. coli-10 region, but both share the quality ofbeingAT-rich element.
Instead of a single bacterial polymerase, the genomes of most eukaryotes encode three different RNA polymerases, each made up of ten protein subunits or more. Each eukaryotic polymerase also requires a distinct set of proteins known astranscription factorsto recruit it to a promoter. In addition, an army of other transcription factors, proteins known as enhancers, and silencers help to regulate the synthesis of RNA from each promoter. Enhancers and silencers affect the efficiency of transcription but arenot necessaryfor the initiation of transcription or its procession. Basal transcription factors are crucial in the formation of apreinitiation complexon the DNA template that subsequently recruits RNA polymerase for transcription initiation.
The initiation of transcription begins with the binding of RNA polymerase to thepromoter. Transcription requires the DNA double helixto partially unwindsuch that one strand canbe used asthe template for RNA synthesis. The region of unwindingis calledatranscription bubble.
Elongation
Transcription always proceeds from thetemplate strand, one of the two strands of the double-stranded DNA. The RNA product is complementary to the template strand and is almost identical to thenontemplatestrand, called thecoding strand, with the exception that RNA contains a uracil (U) in place of the thymine (T) found in DNA. During elongation, an enzyme calledRNA polymeraseproceeds along the DNA template, adding nucleotides by base pairing with the DNA template in a manner similar to DNA replication, with the difference being an RNA strand thatis synthesizeddoes not remain bound to the DNA template. As elongation proceeds, the DNAis continuously unwoundahead of the core enzyme and rewound behind it. Note that the direction of synthesis is identical to that ofsynthesisin DNA—5' to 3'.
Figure 5. During elongation, RNA polymerase tracks along the DNA template, synthesizingmRNAin the 5' to 3' direction, unwinding and then rewinding the DNA asit is read.
Bacterial vs. eukaryotic elongation
In bacteria, elongation begins with the release of theσsubunit from the polymerase. The dissociation ofσallows the core enzyme to proceed along the DNA template, synthesizingmRNAin the 5' to 3' direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNAis continuously unwoundahead of the core enzyme and rewound behind it. The base pairing between DNA and RNA is not stable enough to maintain the stability of themRNAsynthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongationis not interruptedprematurely.
In eukaryotes, following the formation of the preinitiation complex, the polymeraseis releasedfrom the other transcription factors, andelongation is allowedto proceed as it does in prokaryotes with the polymerase synthesizingpre-mRNAin the 5' to 3' direction. As discussed previously, RNA polymerase II transcribes the major share of eukaryotic genes, so this section will focus on how this polymerase accomplishes elongation and termination.
Possible NB Discussion
Point
Compare and contrast the energy story for DNA replication initiation + elongation to the energy story for transcription initiation + elongation.
Termination
In bacteria
Once a geneis transcribed, the bacterial polymerase needs todissociate from the DNA template and liberate the newly mademRNA. Depending on the gene being transcribed, there are two kinds of termination signals. One isaprotein-based, and the other is RNA-based.Rho-dependent terminationis controlledby therhoprotein, which tracks along the polymerase on the growingmRNAchain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, therhoprotein collides with the polymerase. The interaction with rho releases themRNAfrom the transcription bubble.
Rho-independent terminationis controlledby specific sequences in the DNA template strand. As the polymerase nears the end of the gene being transcribed, it encounters a region rich in CG nucleotides. ThemRNAfolds back on itself, and the complementary CG nucleotides bindtogether. The result is a stablehairpinthat causes the polymerase to stall as soon as itbegins to transcribea region rich in AT nucleotides. The complementary UA region of themRNAtranscript 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 newmRNAtranscript.
In eukaryotes
The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place1,000–2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNAtailis subsequently removedby cleavage duringmRNAprocessing.On the other hand, RNApolymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence thatis recognizedby a termination protein. The process of termination in RNA polymerase III involves amRNAhairpin similartorho-independent termination of transcription in prokaryotes.
In archaea
Termination of transcription in the archaea is far less studied than in the other two domains of life andis still not well understood. While the functional details are likely to resemble mechanisms that havebeen seenin the other domains of life, the details are beyondthe scope ofthis course.
Cellular location
In bacteria and archaea
In bacteria and archaea, transcription occurs in thecytoplasm,where the DNAis located. Because the location of the DNA, and thus the process of transcription,are not physically segregatedfrom the rest of the cell, translation often starts before transcription has finished. This means thatmRNAin bacteria andarchaeais usedas the template for a protein before it produces the entiremRNA. The lack of spatial segregation also means that there is very little temporal segregation for these processes. Figure 7 shows the processes of transcription and translation occurring simultaneously.
In eukaryotes....
In eukaryotes, the process of transcriptionis physically segregatedfrom the rest of the cell, sequestered inside of the nucleus. This results in two things:themRNAis completedbefore translation can start, and there is time to "adjust" or "edit" themRNAbefore translation starts. The physical separation of these processes gives eukaryotes a chance to alter themRNAin such a way as to extend the lifespan of themRNAor even alter the protein product that willbe producedfrom themRNA.
mRNAprocessing
5' G-cap and 3' poly-A tail
Whenan eukaryoticgeneis transcribed, the cell processes the primary transcript in the nucleus in several ways. Eukaryotic cellsmodifymRNAsat the 3' end by the addition of a poly-A tail. This run of A residueis addedby an enzyme that does not use genomic DNA as a template. ThemRNAshave a chemical modification of the 5' end, called a 5'-cap. Data suggests that these modifications both help to increase the lifespan of themRNA(prevent its premature degradation in the cytoplasm) and to help themRNAstart translation.
Possible NB DiscussionPoint
Transcriptomics is a branch of “-omics” that involves studying an organism or population’s transcriptome or, the complete set of all RNA molecules. What kind of information can you obtain from studying the transcriptome(s)? Can you think of any cool scientific questions that a transcriptomic analysis might help resolve? What are some constraints to transcriptomic approaches one might keep in mind when conducting analyses?
Alternative splicing
Splicing occurs on most eukaryoticmRNAsin which intronsare removedfrom themRNAsequence and exonsare ligatedtogether. This can create a much shortermRNAthan initially transcribed. Splicing allows cells to mix and matchwhichexonsare incorporatedinto the finalmRNAproduct. As shown in the figure below, this can lead to multiple proteins being coded for by a single gene.
Link to external resources
DNA, Hot Pockets, & The Longest Word Ever: Crash Course Biology #11
https://www.youtube.com/watch?v=itsb2SqR-R0
Transcription and Translation: From DNA to Protein
