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Lecture 13: Reading information in the genome: transcription Translation and post-translational processes

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 are fundamentally the same and can be characterized by three stages: initiation, elongation, and termination.

Transcription: from DNA to RNA

A short overview of transcription

Transcription is the process of creating 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, if anything, catalyzes the process? We also want to think about the process from a Design Challenge standpoint. If the biological task is to create a copy of DNA in the chemical language of RNA, what challenges can we reasonably hypothesize or anticipate, given our knowledge about other nucleotide polymer processes, must be overcome? Is there evidence that Nature solved these problems in different ways? What seem to be the criteria for success of transcription? You get the idea.

Listing some of the 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 in order to properly describe the process.  

Let's imagine that we want to design a nanomachine/nanobot that would conduct transcription. We can use some Design Challenge thinking to identify problems and subproblems that need to be solved by our little robot. 

• Where should the machine start? Along the millions to billions of base pairs, where should the machine be 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 that be accomplished?
• How many RNA copies of the DNA will we need to make? 
• How fast do the RNA copies need to be made?  
• How accurately do the copies need to be made?  
• How much energy will the process take and where is the energy going to come from?  

These are, of course, only some of the core questions. One can dig deeper if they wish. However, these are already good enough for us to start getting 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 about 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 consists of nucleotide triphosphates that are composed of a ribose sugar, a nitrogenous base, and three phosphate groups. The key differences between the building blocks of DNA and those of RNA are that RNA molecules are composed of nucleotides with ribose sugars (as opposed to deoxyribose sugars) and utilize 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


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 to be copied. A promoter is a DNA sequence onto which various proteins, collectively known as the transcription machinery, bind and initiates 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 corresponding coding portion of the gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among species, a few elements of similar sequence are sometimes conserved. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are 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 initiate transcription. This helps to control the number of transcripts that are made and 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. coli promoter sequences. The -35 box and -10 box are highly conserved sequences throughout the strong promoter list. Weaker promoters will have more base pair differences when compared to these sequences.

Note: possible discussion

What types of interactions are changed between the transcription machinery and the DNA when the nucleotide sequence of the promoter changes? Why would some sequences create a "strong" promoter and why do others create a "weak" promoter?

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 recognized and bound by the protein σ. Once this protein-DNA interaction is made, the subunits of the core RNA polymerase bind to the site. Due to 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 is typically called a TATA box. For example, in the mouse thymidine kinase gene, the TATA box is located at approximately -30. 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 being 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 not necessary 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. 

Initiation of transcription begins with the binding of RNA polymerase to the promoter. Transcription requires the DNA double helix to partially unwind such that one strand can be used as the template for RNA synthesis. The region of unwinding is called a transcription bubble.

Figure 3. During elongation, RNA polymerase tracks along the DNA template, synthesizes mRNA in the 5' to 3' direction, and unwinds then rewinds the DNA as it is read.


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 that is synthesized does not remain bound to the DNA template. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. Note that the direction of synthesis is identical to that of synthesis in 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 as it is read.



Figure 5. The addition of nucleotides during the process of transcription is very similar to nucleotide addition in DNA replication. The RNA is polymerized from 5' to 3', and with each addition of a nucleotide, a phosphoanhidride bond is hydrolized by the enzyme, resulting in a longer polymer and the release of two inorganic phosphates.

Note: possible discussion

Compare and contrast the energy story for the addition of a nucleotide in DNA replication to the addition of a nucleotide in transcription.

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 is continuously unwound 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 is not interrupted prematurely. 

In eukaryotes, following the formation of the preinitiation complex, the polymerase is released from the other transcription factors, and elongation is allowed 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.


In bacteria

Once a gene is transcribed, the bacterial 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 CG nucleotides. The mRNA folds back on itself, and the complementary CG 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 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 place 1,000–2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. On the other hand, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence that is recognized by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-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 and is still not well understood. While the functional details are likely to resemble mechanisms that have been seen in the other domains of life, the details are beyond the scope of this course. 

Cellular location

In bacteria and archaea

In bacteria and archaea, transcription occurs in the cytoplasm, where the DNA is located. Because the location of the DNA, and thus the process of transcription, are not physically segregated from 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 the entire mRNA is produced. The lack of spacial segregation also means that there is very little temporal segregation for these processes. Figure 6 shows the processes of transcription and translation occurring simultaneously.

Figure 6. The addition of nucleotides during the process of transcription is very similar to nucleotide addition in DNA replication. 
Source: Marc T. Facciotti (own work)

In eukaryotes....

In eukaryotes, the process of transcription is physically segregated from 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 will be produced from the mRNA.

mRNA processing

5' G-cap and 3' poly-A tail

When a eukaryotic gene is transcribed, the primary transcript is processed in the nucleus in several ways. Eukaryotic mRNAs are modified at the 3' end by the addition of a poly-A tail. This run of A residues is added by an enzyme that does not use genomic DNA as a template. Additionally, 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) as well as to help the mRNA initiate translation.

Figure 7. pre-mRNAs are processed in a series of steps. Introns are removed, a 5' cap and poly-A tail are added.

Alternative splicing

Splicing occurs on most eukaryotic mRNAs in which introns are removed from the mRNA sequence and  exons are ligated together. This can create a much shorter mRNA than initially transcribed. Splicing allows cells to mix and match which exons are incorporated into the final mRNA product. As shown in the figure below, this can lead to multiple proteins being coded for by a single gene.

Figure 8. The information stored in the DNA is finite. In some cases, organisms can mix and match this information to create different end products. In eukaryotes, alternative splicing allows for the creation of different mRNA products, which in turn are used in translation to create different protein sequences. This ultimately leads to the production of different protein shapes, and thus different protein functions. 


Protein Synthesis


The process of translation in biology is the decoding an mRNA message into a polypeptide product. Put another way, a message written in the chemical language of nucleotides is "translated" into the chemical language of amino acids. Amino acids are linearly strung together via covalent bonds (called peptide bonds) between amino and carboxyl termini of adjacent amino acids. The decoding and "linking" process is catalyzed by a ribonucleoprotein complex called the ribosomes and can result in chains of amino acids of lengths ranging from tens to more than 1,000.  

The resulting proteins are so important to the cell that their synthesis consumes more of a cell’s energy than any other metabolic process. Like DNA replication and transcription, translation is a complex molecular process that we can approach using both the Energy Story and Design Challenge rubrics. Describing the overall process, or steps in the process, requires the accounting of the matter and energy before the process and after the process and a description of how that matter is transformed and energy transferred during the process. From a Design Challenge standpoint, we can - even before digging any further into what is or is not understood about translation - try to infer some of the basic questions that we will need to answer regarding this process.  

Let us start by considering the basic problem. We have a strand of RNA (called mRNA) and a bunch of amino acids and we need to somehow design a machine that will: 

(a) decode the chemical language of nucleotides into the language of amino acids, 
(b) join amino acids in a very specific manner, 
(c) complete this process with reasonable accuracy, and 
(d) do this at a reasonable speed. Reasonable, is of course defined by natural selection.  

As before, we can identify subproblems 

(a) How does our molecular machine determine where and when to start working?
(b) How does the molecular machine coordinate decoding and bond formations? 
(c) where does the energy for this process come from and how much?
(d) how does the machine know where to stop?  

Other questions and functional problems/challenges will certainly arise as we dig deeper. 

The point, as always, is that even without knowing any specifics about translation we can use our imaginations, curiosity and common sense to imagine some requirements for the process that we will need to learn more about. Understanding these questions as the context for what follows is key.

A peptide bond links the carboxyl end of one amino acid with the amino end of another, expelling one water molecule. The R1 and R2 designation refer to side chain of amino acid the two amino acids. 
Attribution: Marc T. Facciotti (original work).

Protein Synthesis Machinery

The components that go into the process

Many different molecules and macromolecules contribute to the process of translation. While the exact composition of "the players" in the process may vary from species to species - for instance, ribosomes may consist of different numbers of rRNAs (ribosomal RNAs) and polypeptides depending on the organism - the general functions of the protein synthesis machinery are comparable from bacteria to human cells. We focus on these similarities. At a minimum, translation requires an mRNA templateamino acidsribosomestRNAs, an energy source, and various additional accessory enzymes and small molecules.

Reminder: Amino acids

Let us simply recall that the basic structure of amino acids is composed of a backbone composed of an amino group, a central carbon (called the α-carbon), and a carboxyl group. Attached to the α-carbon is a variable group that helps determine some of the chemical properties and reactivity of the amino acid.

A generic amino acid.
Attribution: Marc T. Facciotti (own work)

The 20 common amino acids.
Attribution: Marc T. Facciotti (own work)


ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. As we start to try thinking about energy accounting in the cell it is worth noting that ribosomes do not come "free". Even before an mRNA is translated, a cell must invest energy to build each of its ribosomes. In E. coli, there are between 10,000 and 70,000 ribosomes present in each cell at any given time. 

Ribosomes exist in the cytoplasm in bacteria and archaea and in the cytoplasm and on the rough endoplasmic reticulum in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to bacterial ribosomes (and have similar drug sensitivities), than the ribosomes just outside their outer membranes in the cytoplasm. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S, and the large subunit is 50S. Mammalian ribosomes have a small 40S subunit and a large 60S subunit. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5' to 3' and synthesizing the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.

The protein synthesis machinery includes the large and small subunits of the ribosome, mRNA, and tRNA.


tRNAs are structural RNA molecules that were transcribed from genes. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. 

Of the 64 possible mRNA codons—or triplet combinations of A, U, G, and C, three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of these 61, one codon (AUG) also encodes the initiation of translation. Each tRNA anticodon can base pair with one of the mRNA codons and add an amino acid or terminate translation, according to the genetic code. For instance, if the sequence CUA occurred on an mRNA template in the proper reading frame, it would bind a tRNA expressing the complementary sequence, GAU, which would be linked to the amino acid leucine.

The folded secondary structure of a tRNA. The anticodon loop and amino acid acceptor stem are indicated. 
Source: http://mol-biol4masters.masters.grkr...ansfer_RNA.htm

Aminoacyl tRNA Synthetases

The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule. The corresponding amino acid must be added later, once the tRNA is processed and exported to the cytoplasm. Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction. The activated amino acid is then transferred to the tRNA, and AMP is released.

The Mechanism of Protein Synthesis

Just as with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in bacteria, archaea and eukaryotes. 

Translation Initiation

In general, protein synthesis begins with the formation of an initiation complex. The small ribosomal subunit will bind to the mRNA at the ribosomal binding site. Soon after, the methionine-tRNA will bind to the AUG start codon (through complementary binding with its anticodon). This complex is then joined by large ribosomal subunit. This initiation complex then recruits the second tRNA and thus translation begins.

Translation begins when a tRNA anticodon recognizes a codon on the mRNA. The large ribosomal subunit joins the small subunit, and a second tRNA is recruited. As the mRNA moves relative to the ribosome, the polypeptide chain is formed. Entry of a release factor into the A site terminates translation and the components dissociate.

Bacterial vs Eukaryotic initiation

In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with a rRNA molecule. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. Stop for a moment to appreciate the repetition of a mechanism you've encountered before. In this case, getting a protein complex to associate - in proper register - with a nucleic acid polymer is accomplished by aligning two antiparallel strands of complementary nucleotides with one another. We also saw this in the function of telomerase.  

Instead of binding at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA. A cap-binding protein (CBP) assists the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex tracks along the mRNA in the 5' to 3' direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak’s rules, the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-gccRccAUGG-3'. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Translation Elongation

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into 'view', and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically. Note again the use of base pairing between two antiparallel strands of complementary nucleotides to bring and keep our molecular machine in register and in this case also to accomplish the job of "translating" between the language of nucleotides and amino acids.  

The large ribosomal subunit consists of three compartments: the A site binds incoming charged tRNAs (tRNAs with their attached specific amino acids), the P site binds charged tRNAs carrying amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA, and the E site which releases dissociated tRNAs so they can be recharged with another free amino acid. 

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3' direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. 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 enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. 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. The ribosome moves along the mRNA, one codon at a time, catalyzing each process that occurs in the three sites. With each step, a charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs. Amazingly, this process occurs rapidly in the cell, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid polypeptide could be translated in just 10 seconds.

Suggested discussion

Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?

The Genetic Code

To summarize what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon. The relationship between a nucleotide codon and its corresponding amino acid is called the genetic code. Given the different numbers of “letters” in the mRNA and protein “alphabets,” means that there are a total of 64 (4 × 4 × 4) possible codons; therefore, a given amino acid (20 total) must be encoded for by more than one codon. 

Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.

This figure shows the genetic code for translating each nucleotide triplet, or codon, in mRNA into an amino acid or a termination signal in a nascent protein. (credit: modification of work by NIH)

Redundant, not Ambiguous

The information in the genetic code is redundant. Multiple codons code for the same amino acid. For example, using the chart above, you can find 4 different codons that code for Valine, likewise, there are two codons that code for Leucine, etc. But the code is not ambiguous, meaning, that if you were given a codon you would know definitively which amino acid it is coding for, a codon will only code for a specific amino acid. For example, GUU will always code for Valine, and AUG will always code for Methionine. This is important, you will be asked to translate an mRNA into a protein using a codon chart like the one shown above.

Translation Termination

Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome encounters the stop codon no tRNA enters into the A site. Instead a protein know as a release factor binds to the complex. This interaction destabilizes the translation machinery, causing the release of the polypeptide and the dissociation of the ribosome subunits from the mRNA. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Suggested discussion

What are the benefits and drawbacks to translating a single mRNA multiple times?

Coupling between Transcription and Translation

As discussed previously, bacteria and archaea do not need to transport their RNA transcripts between a membrane bound nucleous and the cytoplasm. The RNA polymerase is therefore transcribing RNA directly into the cytoplasm. Here ribosomes can bind to the RNA and begin the process of translation, in some instances while transciption is still occurring. The coupling of these two processes, and even mRNA degradation, is facilitated not only because transcription and translation happen in the same compartment but also because both of the processes happen in the same direction - synthesis of the RNA transcript happens in the 5' to 3' direction and translation reads the transcript in the 5' to 3' direction. This "coupling" of transcription with translation occurs in both bacteria and archaea and is, in fact, essential for proper gene expression in some instances.

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.

Protein Sorting

In context of a protein synthesis Design Challenge we can also raise the question/problem of how proteins get to where they are supposed to go. We know that some proteins are destined for the plasma membrane, others in eukaryotic cells need to be directed to various organelles, some proteins, like hormones or nutrient scavenging proteins, are intended to be secreted by cells while others may need to be directed to parts of the cytosol to serve structural roles. How does this happen?  

Since various mechanisms have been uncovered, the details of this process are not easily summarized in a brief paragraph or two. However, some key common elements of all mechanisms can be mentioned. First, is the need for a specific "tag" that can provide some molecular information about where the protein of interest is destined. This tag usually takes the form of a short string of amino acids - a so called signal peptide - that can encode information about where the protein is intended to end up. The second required component of the protein sorting machinery must be a system to actually read and sort the proteins. In bacterial and archaeal systems this usually consists of proteins that can identify the signal peptide during translation, bind to it, and direct the synthesis of the nascent protein to the plasma membrane. In eukaryotic systems, the sorting is by necessity more complex, and involves a rather elaborate set of mechanisms of signal recognition, protein modification, and trafficking of vesicles between organelles or the membrane. These biochemical steps are initiated in the endoplasmic reticulum and further "refined" in the Golgi apparatus where proteins are modified and packaged into vesicles bound for various parts of the cell.  

Some of the various specific mechanisms may be discussed by your instructor in class. The key for all students it so appreciate the problem and to have a general idea of the high-level requirements that cells have adopted to solve them. 

Post-translational Protein Modification

After translation individual amino acids may be chemically modified. These modifications add chemical variation and new properties that are rooted in the chemistries of the functional groups that are being added. Common modifications include phosphate groups, methyl, acetate, and amide groups. Some proteins, typically targeted to membranes will be lipidated - a lipid will be added. Other proteins will be glycosylated - a sugar will be added. Another common post-translational modification is cleavage or linking of parts of the protein itself. Signal-peptides may be cleaved, parts may be excised from the middle of the protein, or new covalent linkages may be made between cysteine or other amino acid side chains. Nearly all modifications will be catalyzed by enzymes and all change the functional behavior of the protein.  

Section Summary

mRNA is used to synthesize proteins by the process of translation. The genetic code is the correspondence between the three-nucleotide mRNA codon and an amino acid. The genetic code is “translated” by the tRNA molecules, which associate a specific codon with a specific amino acid. The genetic code is degenerate because 64 triplet codons in mRNA specify only 20 amino acids and three stop codons. This means that more than one codon corresponds to an amino acid. Almost every species on the planet uses the same genetic code. 

The players in translation include the mRNA template, ribosomes, tRNAs, and various enzymatic factors. The small ribosomal subunit binds to the mRNA template. Translation begins at the initiating AUG on the mRNA. The formation of bonds occurs between sequential amino acids specified by the mRNA template according to the genetic code. The ribosome accepts charged tRNAs, and as it steps along the mRNA, it catalyzes bonding between the new amino acid and the end of the growing polypeptide. The entire mRNA is translated in three-nucleotide “steps” of the ribosome. When a stop codon is encountered, a release factor binds and dissociates the components and frees the new protein.