2020_Winter_Bis2a_Facciotti_Lecture_21 - Biology

Figure 3. A geneconsists ofa coding region for an RNA or protein product accompanied by its regulatory regions.The coding region is transcribedintoRNAwhichis then translatedinto protein.

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 a process, 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 know

to describe the process properly.

Let us imagine that we want to design a nanomachine/nanobot that would conduct transcription. We can use some

thinking to identify problems and subproblems that need to

by our little robot.

• Where should the machine start? Along the millions to billions of base pairs, where should the machine

?
• 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 that

?
• How many RNA copies of the DNA will we need to make?
• How fast do the RNA copies need to

?
• How accurately do the copies need to

?
• 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 1. 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. A promoter is a DNA sequence onto which various proteins, collectively known as the transcription machinery, bind and start transcription. 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 twopromoterconsensus sequences, 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 2. (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,

and bound by the protein σ. Once this protein-DNA interaction

, 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, and several phosphodiester bonds are made.

Eukaryotic promoters are much larger and more complex than prokaryotic promoters, but both have an AT-rich region—in eukaryotes, it

a TATA box. For example, in the mouse thymidine kinase gene,

at

. For this gene, the exact TATA box sequence is TATAAAA, as read in the 5' to 3' direction on the nontemplate strand. This sequence is not identical to the E. coli -10 region, but both share the quality of

AT-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 as transcription factors to 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 are

for the initiation of transcription or its procession. Basal transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase for transcription initiation.

The initiation of transcription begins with the binding of RNA polymerase to the promoter. Transcription requires the DNA double helixto partially unwindsuch that one strand canbe used asthe template for RNA synthesis. The region of unwindingis calleda transcription bubble.

Elongation

Transcription always proceeds from the template 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 the nontemplate strand, called the coding strand, with the exception that RNA contains a uracil (U) in place of the thymine (T) found in DNA. During elongation, an enzyme called RNA polymerase proceeds 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 4. During elongation, RNA polymerase tracks along the DNA template, synthesizing mRNA in 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 the σ subunit from the polymerase. The dissociation of σ 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

ahead of the core enzyme and rewound behind it. 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

prematurely.

In eukaryotes, following the formation of the preinitiation complex, the polymerase

from the other transcription factors, and

to proceed as it does in prokaryotes with the polymerase synthesizing pre-mRNA in 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 gene

, the bacterial polymerase needs 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

by the

protein, which tracks along 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

protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble.

Rho-independent termination

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 CG nucleotides. The mRNA folds back on itself, and the complementary CG nucleotides bind

. The result is a stable hairpin that causes the polymerase to stall as soon as it

a region rich in AT nucleotides. The complementary UA 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.

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 during mRNA processing.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 a mRNA hairpin 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 that mRNA in bacteria and archaea is used as the template for a protein before it produces the entire mRNA. The lack of spatial segregation also means that there is very little temporal segregation for these processes. Figure 6 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: the mRNA is completed before translation can start, and there is time to "adjust" or "edit" the mRNA before translation starts. The physical separation of these processes gives eukaryotes a chance to alter the mRNA in such a way as to extend the lifespan of the mRNA or even alter the protein product that willbe producedfrom the mRNA.

MRNA processing

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. The mRNAs have a chemical modification of the 5' end, called a 5'-cap. Data suggests that these modifications both help to increase the lifespan of the mRNA (prevent its premature degradation in the cytoplasm) and to help the mRNA start translation.

Possible NB Discussion Point

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 eukaryotic mRNAs in which intronsare removedfrom the mRNA sequence and exonsare ligatedtogether. This can create a much shorter mRNA than initially transcribed. Splicing allows cells to mix and matchwhichexonsare incorporatedinto the final mRNA product. As shown in the figure below, this can lead to multiple proteins being coded for by a single gene.