An in-depth look how polypeptides (proteins) are made. Initiation, elongation, and termination.

Introduction

Ever wonder how antibiotics kill bacteria—for instance, when you have a sinus infection? Different antibiotics work in different ways, but some attack a very basic process in bacterial cells: they knock out the ability to make new proteins.
To use a little molecular biology vocab, these antibiotics block translation. In the process of translation, a cell reads information from a molecule called a messenger RNA (mRNA) and uses this information to build a protein. Translation is happening constantly in a normal bacterial cell, just like it is in most of the cells of your body, and it's key to keeping you (and your bacterial "visitors") alive.
When you take certain antibiotics (e.g., erythromycin), the antibiotic molecule will latch onto key translation molecules inside of bacterial cells and basically "stall" them. With no way to make proteins, the bacteria will stop functioning and, eventually, die. That's why infections clear up when they're treated with the antibiotic.1,2^{1,2}
Great question! Erythromycin blocks translation only in bacteria, not in humans like you and me. To see why, we need to know a little more about how the antibiotic works.
The erythromycin molecule attaches to a specific site on a key translation molecule, the ribosome. This site is different between humans and bacteria, so the erythromycin can only attach to the bacterial version (and not to the human version).3^3
If this weren't the case, erythromycin would be harmful to us as well as to bacteria!
Cells need translation to stay alive, and understanding how it works (so we can shut it down with antibiotics) can save us from bacterial infections. Let's take a closer look at how translation happens, from the first step to the final product.

Translation: The big picture

Translation involves “decoding” a messenger RNA (mRNA) and using its information to build a polypeptide, or chain of amino acids. For most purposes, a polypeptide is basically just a protein (with the technical difference being that some large proteins are made up of several polypeptide chains).

The genetic code

In an mRNA, the instructions for building a polypeptide come in groups of three nucleotides called codons. Here are some key features of codons to keep in mind as we move forward:
  • There are 6161 different codons for amino acids
  • Three “stop” codons mark the polypeptide as finished
  • One codon, AUG, is a “start” signal to kick off translation (it also specifies the amino acid methionine)
These relationships between mRNA codons and amino acids are known as the genetic code (which you can explore further in the genetic code article).
Genetic code table. Each three-letter sequence of mRNA nucleotides corresponds to a specific amino acid, or to a stop codon. UGA, UAA, and UAG are stop codons. AUG is the codon for methionine, and is also the start codon.
_Image credit: "The genetic code," by OpenStax College, Biology (CC BY 3.0)._

Codons to amino acids

In translation, the codons of an mRNA are read in order (from the 5' end to the 3' end) by molecules called transfer RNAs, or tRNAs.
Each tRNA has an anticodon, a set of three nucleotides that binds to a matching mRNA codon through base pairing. The other end of the tRNA carries the amino acid that's specified by the codon.
Great questions! Let's start with base pairing.
Base pairs happen when bonds form between the nitrogenous bases in DNA and/or RNA molecules. (As a refresher, bases are the chemical groups that are abbreviated A, T, C, and G in DNA, or A, U, C, and G in RNA). Only certain bases can pair with each other: for instance, in typical cases, A bonds with U in RNA molecules.
An example of a tRNA forming base pairs with an mRNA is shown below. You can see three base pairs, each marked with a line:
Image showing a tRNA acting as an adapter connecting an mRNA codon to an amino acid. At one end, the tRNA has an anticodon of 3'-UAC-5', and it binds to a codon in an mRNA that has a sequence of 5'-AUG-3' through complementary base pairing. The other end of the tRNA carries the amino acid methionine (Met), which is the the amino acid specified by the mRNA codon AUG.
Image modified from "Translation: Figure 3," by OpenStax College, Biology (CC BY 4.0).
Now, what about 5' and 3' ends? The basic idea here is that the two ends of a strand of DNA or RNA are different from each other.
  • At the 5’ end of the chain, the phosphate group of the first nucleotide in the chain sticks out.
  • At the other end, called the 3’ end, the hydroxyl of the last nucleotide added to the chain is exposed.
Often, molecular processes can only take place in a certain direction along a DNA or RNA strand. For instance, in translation, the mRNA is always read from the 5' end towards the 3' end.
Curious about why this 5 'and 3' stuff matters? You can learn more about its importance in the article on nucleic acids.
The ribosome provides where an mRNA can interact with tRNAs bearing amino acids. There are three places on the ribosome where tRNAs bind: the A, P, and E site. The A site accepts an incoming tRNA bound to an amino acid. The P site holds a tRNA that carries a growing polypeptide (the first amino acid added is methionine (Met)). The E site is where a tRNA goes after it is empty, meaning that it has transferred its polypeptide to another tRNA (which now occupies the P site). In the diagram, the empty tRNA has already left the E site and is thus not shown.
Image modified from "Translation: Figure 3," by OpenStax College, Biology (CC BY 4.0).
tRNAs bind to mRNAs inside of a protein-and-RNA structure called the ribosome. As tRNAs enter slots in the ribosome and bind to codons, their amino acids are linked to the growing polypeptide chain in a chemical reaction. The end result is a polypeptide whose amino acid sequence mirrors the sequence of codons in the mRNA.
The mRNA sequence is:
5'-AUGAUCUCGUAA-5'
Translation involves reading the mRNA nucleotides in groups of three; each group specifies an amino acid (or provides a stop signal indicating that translation is finished).
3'-AUG AUC UCG UAA-5'
AUG \rightarrow Methionine AUC \rightarrow Isoleucine UCG \rightarrow Serine UAA \rightarrow "Stop"
Polypeptide sequence: (N-terminus) Methionine-Isoleucine-Serine (C-terminus)
That's big picture of translation. But what about the nitty gritty of how translation begins, proceeds, and finishes? Let's take a look.

Translation: Beginning, middle, and end

A book or movie has three basic parts: a beginning, middle, and end. Translation has pretty much the same three parts, but they have fancier names: initiation, elongation, and termination.
  • Initiation ("beginning"): in this stage, the ribosome gets together with the mRNA and the first tRNA so translation can begin.
  • Elongation ("middle"): in this stage, amino acids are brought to the ribosome by tRNAs and linked together to form a chain.
  • Termination ("end"): in the last stage, the finished polypeptide is released to go and do its job in the cell.
Let’s take a closer look at how each stage works.

Initiation

In order for translation to start, we need a few key ingredients. These include:
  • A ribosome (which comes in two pieces, large and small)
  • An mRNA with instructions for the protein we'll build
  • An "initiator" tRNA carrying the first amino acid in the protein, which is almost always methionine (Met)
During initiation, these pieces must come together in just the right way. Together, they form the initiation complex, the molecular setup needed to start making a new protein.
No, not exactly! Initiation calls for some helper molecules, as well as an energy source.4,5^{4,5}
Initiation depends on specialized protein "helpers" called initiation factors. Their job is to help the ribosome subunits, tRNA, and mRNA find each other in an orderly and predictable way.
Also, there's no such thing as a free lunch: moving those initiation ingredients around requires energy. The energy is provided by the cell in the form of guanosine triphosphate (GTP), a common "energy currency" molecule that's similar to the better-known ATP.
Inside your cells (and the cells of other eukaryotes), translation initiation goes like this: first, the tRNA carrying methionine attaches to the small ribosomal subunit. Together, they bind to the 5' end of the mRNA by recognizing the 5' GTP cap (added during processing in the nucleus). Then, they "walk" along the mRNA in the 3' direction, stopping when they reach the start codon (often, but not always, the first AUG).6^6
Eukaryotic translation initiation:
  1. Complex of small ribosomal subnit and initiator tRNA (bearing methionine) binds to 5' cap of mRNA.
  2. Complex scans from 5' to 3' to find the start codon (AUG).
  3. Initiator tRNA binds to start codon.
  4. Large ribosomal subunit comes together with the mRNA, initiator tRNA, and small ribosomal subunit to form the initiation complex. The initiator tRNA is positioned in the P site of the assembled ribosome.
These steps are assisted by initiation factors (not shown in diagram).
Based on similar diagram in Berg et al.1^1
In bacteria, the situation is a little different. Here, the small ribosomal subunit doesn't start at the 5' end of the mRNA and travel toward the 3' end. Instead, it attaches directly to certain sequences in the mRNA. These Shine-Dalgarno sequences come just before start codons and "point them out" to the ribosome.
Bacterial translation initiation:
On a bacterial mRNA, a G/A-rich sequence called the Shine-Dalgarno sequence is found slightly upstream (5' of) the start codon (AUG). The small ribosomal subunit recognizes and binds to the Shine-Dalgarno sequence. The small ribosomal subunit also binds to the initiator tRNA (carrying fMet), which forms complementary base pairs with the start codon. As noted in the text, the small ribosomal subunit may sometimes bind first to the mRNA (and then the initiator tRNA), and sometimes the other way around (the initiator tRNA first, and then the mRNA). It's thought that the order of these events may be random.
Once these components have come together, the large ribosomal subunit joins them. The assembled ribosome with mRNA and bound initiator tRNA comprises the initiation complex. The initiator tRNA is in the P site of the assembled ribosome.
Bacteria use fMet (a chemically modified methionine) as the first amino acid.
Why use Shine-Dalgarno sequences? Bacterial genes are often transcribed in groups (called operons), so one bacterial mRNA can contain the coding sequences for several genes. A Shine-Dalgarno sequence marks the start of each coding sequence, letting the ribosome find the right start codon for each gene.
Eukaryotic cell:
  • DNA is transcribed to make an RNA inside the nucleus. The initial RNA transcript is processed into a mature mRNA before exportation to the cytosol.
  • The mRNA contains just one coding sequence (specifying one polypeptide).
Bacterial cell:
  • DNA is transcribed to make an mRNA in the cytosol. Ribosomes can start translating the mRNA before it is even completely transcribed. No post-transcription processing steps are necessary.
  • The mRNA contains three coding sequences from three different genes, each specifying its own polypeptide.

Elongation

I like to remember what happens in this "middle" stage of translation by its handy name: elongation is when the polypeptide chain gets longer.
But how does the chain actually grow? To find out, let's take a look at the first round of elongation—after the initiation complex has formed, but before any amino acids have been linked to make a chain.
Our first, methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site. Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be the "landing site" for the next tRNA, one whose anticodon is a perfect (complementary) match for the exposed codon.
Non-matching tRNAs may also enter the A site. Why doesn't this cause wrong amino acids to be added to the chain?
Well, non-matching tRNAs may be able to enter the A site...but in general, they don't get to stay there. In a careful process that never fails to fascinate me, each tRNA is escorted by helper proteins, and only a tRNA that's a perfect match for the codon will be "released" into the A slot by its choosy helpers.7^7
A molecule of the energy storage molecule guanosine triphosphate (GTP) is used up to release the tRNA, which is why the diagram shows this step as needing GTP.
In the first round of elongation, an incoming amino acid attaches to methionine already present in the ribosome's P site. This action initiates the growth of a polypeptide. The three steps of this first round of elongation are described below.
1) Codon recognition: an incoming tRNA with an anticodon that is complementary to the codon exposed in the A site binds to the mRNA. Energy from GTP is expended to increase the accuracy of codon recognition.
2) Peptide bond formation: a peptide bond is formed between the incoming amino acid (carried by a tRNA in the A site) and methionine (a tRNA charged with methionine attached to the P site during initiation). This action passes the polypeptide (the two bonded amino acids) from the tRNA in the P site to the tRNA in the A site. The tRNA in the P site is now "empty" because it does not hold the polypeptide.
3) Translocation: the ribosome moves one codon over on the mRNA toward the 3' end. This shifts the tRNA in the A site to the P site, and the tRNA in the P site to the E site. The empty tRNA in the E site then exits the ribosome.
Once the matching tRNA has landed in the A site, it's time for the action: that is, the formation of the peptide bond that connects one amino acid to another. This step transfers the methionine from the first tRNA onto the amino acid of the second tRNA in the A site.
Not bad—we now have two amino acids, a (very tiny) polypeptide! The methionine forms the N-terminus of the polypeptide, and the other amino acid is the C-terminus.
Great question! Amino acids have an amino group (NH2-\text{NH}_2) at one end and a carboxyl group (COOH-\text{COOH}) at the other:
Diagram of an amino acid, showing the amino and carboxyl groups. Both the amino group and the carboxyl group are attached to a central carbon (the alpha carbon), as is the R group, a chemical group that varies among the different amino acids.
The amino acid is shown in the form it would typically take under physiological conditions, meaning that the amino group is protonated (NH3+-\text{NH}_3^+) and the carboxyl group is deprotonated (COO-COO^-).
A polypeptide is made up of amino acids connected in a chain. Although the amino and carboxyl groups of most amino acids in the chain will be tied up in peptide bonds, the amino acids at the very ends of the chain will each have a free chemical group.
  • One end of the polypeptide has an exposed amino group. This end is called the N-terminus, for the nitrogen atom of the amino group.
  • The other end of the polypeptide has an exposed carboxyl group. This end is called the C-terminus, for the carbon atom of the carboxyl group.
Linear chain of amino acids (polypeptide) with the N and C termini marked. The N terminus has an exposed amino group. The C terminus has an exposed carboxyl group.
The first amino acid in a polypeptide (the methionine carried by the first tRNA) lies at the N-terminus, and new amino acids are progressively added at the C-terminus:
Peptide bond formation. The carboxyl group exposed at the C terminus of a polypeptide reacts with the amino group of an incoming amino acid, forming a peptide bond. This reaction adds the new amino acid to the chain. The carboxyl group of the newly added amino acid becomes the new C terminus of the polypeptide.
But...odds are we may want a longer polypeptide than two amino acids. How does the chain continue to grow? Once the peptide bond is formed, the mRNA is pulled onward through the ribosome by exactly one codon. This shift allows the first, empty tRNA to drift out via the E ("exit") site. It also exposes a new codon in the A site, so the whole cycle can repeat.
And repeat it does...from a few times up to a mind-boggling 33,33,000000 times! The protein titin, which is found in your muscles and is the longest known polypeptide, can have up to 33,33,000000 amino acids8,9^{8,9}.

Termination

Polypeptides, like all good things, must eventually come to an end. Translation ends in a process called termination. Termination happens when a stop codon in the mRNA (UAA, UAG, or UGA) enters the A site.
Stop codons are recognized by proteins called release factors, which fit neatly into the P site (though they aren't tRNAs). Release factors mess with the enzyme that normally forms peptide bonds: they make it add a water molecule to the last amino acid of the chain. This reaction separates the chain from the tRNA, and the newly made protein is released.
What next? Luckily, translation "equipment" is very reusable. After the small and large ribosomal subunits separate from the mRNA and from each other, each element can (and usually quickly does) take part in another round of translation.

Epilogue: Processing

Our polypeptide now has all its amino acids—does that mean it's ready to to its job in the cell?
Not necessarily. Polypeptides often need some "edits." During and after translation, amino acids may be chemically altered or removed. The new polypeptide will also fold into a distinct 3D structure, and may join with other polypeptides to make a multi-part protein.
Many proteins are good at folding on their own, but some need helpers ("chaperones") to keep them from sticking together incorrectly during the complex process of folding.
Some proteins also contain special amino acid sequences that direct them to certain parts of the cell. These sequences, often found close to the N- or C-terminus, can be thought of as the protein’s “train ticket” to its final destination. For more about how this works, see the article on protein targeting.

Attribution

This article is a modified derivative of "Ribosomes and protein synthesis," by OpenStax College, Biology, CC BY 4.0. Download the original article for free at http://cnx.org/contents/185cbf87-c72e-48f5-b51e-f14f21b5eabd@10.59.
The modified article is licensed under a CC BY-NC-SA 4.0 license

Works cited

  1. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). Eukaryotic protein synthesis differs from prokaryotic protein synthesis primarily in translation initiation. In Biochemistry. (5th ed., section 29.5.1). New York, NY: W. H. Freeman. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK22531/#_A4206_.
  2. Purves, W.K., Sadava, D., Orians, G.H., and Heller, H.C. (2004). From DNA to protein: Genotype to phenotype. In Life: The science of biology (7th ed., pp. 246-247). Sunderland, MA: Sinauer Associates, Inc.
  3. Ophardt, Charles E. (2003). Other antibiotics. In Virtual Chembook. Retrieved October 22, 2016 from http://chemistry.elmhurst.edu/vchembook/654antibiotic.html.
  4. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). Protein factors play key roles in protein synthesis. In Biochemistry. (5th ed., section 29.4.1). New York, NY: W. H. Freeman. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK22408/#_A4190_.
  5. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). Eukaryotic protein synthesis differs from prokaryotic protein synthesis primarily in translation initiation. In Biochemistry. (5th ed., section 29.5). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22531/.
  6. Marintchev, A. and Wagner, G. (2004). Translation initiation: Structures, mechanisms, and evolution. In Quarterly Reviews of Biophysics, 37(3/4), 214-215. http://dx.doi.org/10.1017/S0033583505004026. Retrieved from https://gwagner.med.harvard.edu/sites/gwagner.med.harvard.edu/files/marintchev_qrb.pdf.
  7. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). Protein factors play key roles in protein synthesis. In Biochemistry. (5th ed., section 29.4.2). New York, NY: W. H. Freeman. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK22408/#_A4192_
  8. Titin. (2016, October 12). Retrieved October 22, 2016 from Wikipedia: https://en.wikipedia.org/wiki/Titin.
  9. Opitz, C. A., Kulke, M., Leake, M. C., Neagoe, C., Hinssen, H., Hajjar, R. J., and Linke, W. A. (2003). Damped elastic recoil of the titin spring in myofibrils of human myocardium. PNAS, 100(22), 12688. http://dx.doi.org/10.1073/pnas.2133733100.

References

Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). A ribosome is a ribonucleoprotein particle (70S) made of a small (30S) and a large (50S) subunit. In Biochemistry. (5th ed., section 29.2). New York, NY: W. H. Freeman. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK22335/.
Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). Eukaryotic protein synthesis differs from prokaryotic protein synthesis primarily in translation initiation. In Biochemistry. (5th ed., section 29.5). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22531/.
Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). Protein factors play key roles in protein synthesis. In Biochemistry. (5th ed., section 29.4). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22408/.
Cooper, G. M. (2000). Translation of mRNA. In The cell: A molecular approach. (2nd ed.). Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK9849/.
Erythromycin. (2010, September 1). In MedlinePlus. Retrieved from https://www.nlm.nih.gov/medlineplus/druginfo/meds/a682381.html.
Erythromycin. (2015, December 27). Retrieved December 31, 2015 from Wikipedia: https://en.wikipedia.org/wiki/Erythromycin.
Eukaryotes use a complex of many initiation factors. (2007). In Protein synthesis. Retrieved from http://bioscience.jbpub.com/cells/MBIO269.aspx.
Initiation involves base pairing between mRNA and rRNA. (2007). In Protein synthesis. Retrieved from http://bioscience.jbpub.com/cells/MBIO267.aspx.
Initiator tRNA. (n.d). A special initiator tRNA starts the polypeptide chain. Retrieved December 31, 2015 from http://molecularstudy.blogspot.com/2012/10/a-special-initiator-trna-starts.html.
Laursen, B. S., Sørensen, H. P., Mortensen, K. K., and Sperling-Petersen, H. U. (2005). Initiation of protein synthesis in bacteria. Microbiol. Mol. Biol. Rev., 69(1), 101-123. http://dx.doi.org/10.1128/MMBR.69.1.101-123.2005.
Marintchev, A. and Wagner, G. (2004). Translation initiation: Structures, mechanisms, and evolution. In Quarterly Reviews of Biophysics, 37(3/4), 197-284. http://dx.doi.org/10.1017/S0033583505004026. Retrieved from https://gwagner.med.harvard.edu/sites/gwagner.med.harvard.edu/files/marintchev_qrb.pdf.
Nakagawa, S., Niimura, Y., Miura, K., and Gojobori, T. (2010). Dynamic evolution of translation initiation mechanisms in prokaryotes. PNAS, 107(14), 6382-6387. http://dx.doi.org/10.1073/pnas.1002036107.
Neyfakh, A. and Mankin, A. (2000, January 30). Fundamentals of drug action I: lecture 3. Retrieved from http://www.uic.edu/classes/phar/phar331/.
N-Formylmethionine. (2015, December 13). Retrieved December 21, 2015 from Wikipedia: https://en.wikipedia.org/wiki/N-Formylmethionine.
OpenStax College, Biology. (2015, September 30). Ribosomes and protein synthesis. In OpenStax CNX. Retrieved from https://cnx.org/contents/s8Hh0oOc@8.57:FUH9XUkW@6/Translation.
OpenStax College, Biology. (n.d.). Translation. In OpenStax CNX. Retrieved from http://philschatz.com/biology-concepts-book/contents/m45479.html.
Ophardt, Charles E. (2003). Other antibiotics. In Virtual Chembook. Retrieved October 22, 2016 from http://chemistry.elmhurst.edu/vchembook/654antibiotic.html.
Opitz, C. A., Kulke, M., Leake, M. C., Neagoe, C., Hinssen, H., Hajjar, R. J., and Linke, W. A. (2003). Damped elastic recoil of the titin spring in myofibrils of human myocardium. PNAS, 100(22), 12688-12693. http://dx.doi.org/10.1073/pnas.2133733100.
Purves, W.K., Sadava, D., Orians, G.H., and Heller, H.C. (2004). From DNA to protein: Genotype to phenotype. In Life: The science of biology (7th ed., pp. 233-256). Sunderland, MA: Sinauer Associates, Inc.
Rasmussen, L. C. V., Laursen, B. S., Mortensen, K. K., and Sperling-Petersen, H. U. (2009). Initiator tRNAs in bacteria and eukaryotes. eLS. http://dx.doi.org/10.1002/9780470015902.a0000543.pub2.
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). Translation is the RNA-directed synthesis of a polypeptide: A closer look. In Campbell biology (10th ed., pp. 345-353). San Francisco, CA: Pearson.
Small subunits scan for initiation sites on eukaryotic mRNA. (2007). In Protein synthesis. Retrieved from http://bioscience.jbpub.com/cells/MBIO268.aspx.
Titin. (2016, October 12). Retrieved October 22, 2016 from Wikipedia: https://en.wikipedia.org/wiki/Titin.
Loading