Where Does Translation Happen? Unraveling Protein Synthesis in Cells

How do cells transform the genetic code within DNA into functional proteins? The translation process acts as a crucial decoder, converting the instructions for protein synthesis, which are transcribed onto messenger RNA (mRNA), into the amino acid sequences of proteins with the help of transfer RNA (tRNA).

Genes within DNA contain the blueprints for protein molecules, the cell’s essential “workhorses.” These proteins execute life’s critical functions. Enzymes, for instance, are proteins that drive metabolism and synthesize cellular components. DNA polymerases, vital for DNA replication during cell division, are also proteins.

Gene expression, in its simplest form, is the creation of a corresponding protein through a two-step process. First, transcription transfers genetic information from DNA to an mRNA molecule. During transcription, a gene’s DNA acts as a template for complementary base pairing, and RNA polymerase II, an enzyme, facilitates the creation of a pre-mRNA molecule. This precursor is then refined into mature mRNA (Figure 1). The resulting mRNA is a single-stranded gene copy, ready for the next step: translation into a protein.

Figure 1: Gene Expression: Transcription and Translation.

Transcription, depicted here, shows RNA polymerase (in green) using a DNA template to synthesize a pre-mRNA transcript (in pink). This pre-mRNA is then processed into a mature mRNA molecule, which serves as the blueprint for translation, ultimately leading to the protein molecule (polypeptide) encoded by the original gene.

During translation, the second key step in gene expression, mRNA is “read” according to the genetic code. This code correlates DNA sequences with protein amino acid sequences (Figure 2). Every three-base group in mRNA is a codon, specifying a particular amino acid – hence, a triplet code. The mRNA sequence guides the assembly of amino acids in a precise order, forming the protein chain.

Figure 2: mRNA Codons and Corresponding Amino Acids.

This table illustrates the amino acids specified by each mRNA codon. Notice that multiple codons can code for the same amino acid, showing redundancy in the genetic code. AUG serves as the initiation codon, while UAA, UAG, and UGA are termination (stop) codons, signaling the end of protein synthesis. Codons are read from 5′ to 3′ as they appear in the mRNA sequence.

But Where Does Translation Happen within the cell? What are the specific steps involved? And does translation differ between prokaryotic and eukaryotic cells? Exploring these questions unveils fundamental similarities across all life forms.

The Cellular Location of Translation: Ribosomes

Within all cells, the machinery for translation is housed in ribosomes, specialized organelles. In eukaryotes, mature mRNA molecules must exit the nucleus and enter the cytoplasm, where ribosomes reside. Conversely, in prokaryotes, ribosomes can attach to mRNA while transcription is still ongoing. Here, translation starts at the 5′ end of the mRNA, even as the 3′ end remains linked to DNA.

Regardless of cell type, a ribosome comprises two subunits: a large (50S) and a small (30S) subunit (Svedberg units, S, measure sedimentation velocity and thus mass). These subunits exist separately in the cytoplasm but unite on the mRNA molecule to initiate translation. Ribosomal subunits are made of proteins and specialized RNA molecules – ribosomal RNA (rRNA) and transfer RNA (tRNA). tRNA molecules are adaptors: one end recognizes the mRNA triplet code via complementary base pairing, while the other binds to a specific amino acid (Chapeville et al., 1962; Grunberger et al., 1969). Francis Crick, a co-discoverer of DNA structure, first proposed the tRNA adaptor concept, contributing significantly to deciphering the genetic code (Crick, 1958).

Inside the ribosome, mRNA and aminoacyl-tRNA complexes are held in close proximity, facilitating codon-anticodon base pairing. The rRNA catalyzes the crucial step of linking each new amino acid to the growing polypeptide chain.

Untranslated Regions: The Beginning of mRNA

Intriguingly, not all mRNA regions directly translate into amino acids. A segment near the 5′ end, known as the untranslated region (UTR) or leader sequence, exists between the first transcribed nucleotide and the start codon (AUG) of the coding region. This UTR does not alter the protein’s amino acid sequence (Figure 3).

What is the UTR’s function? This leader sequence is vital as it contains a ribosome-binding site. In bacteria, this is the Shine-Dalgarno box (AGGAGG), named after John Shine and Lynn Dalgarno, who identified it. A similar site in vertebrates, the Kozak box, was characterized by Marilyn Kozak. Bacterial mRNA typically has a short 5′ UTR, while human mRNA 5′ UTRs average around 170 nucleotides. Longer leaders may house regulatory sequences, including protein-binding sites, which can affect mRNA stability or translation efficiency.

Figure 3: DNA Transcription Unit Layout.

This diagram shows a DNA transcription unit composed (3′ to 5′) of a pink RNA-coding region, a green promoter region, and a black terminator region. Regions upstream (towards 3′ end) of the transcription start site are “upstream,” and regions downstream (towards 5′ end) are “downstream.”

Initiation Complex: Starting Translation

Translation initiation begins with the formation of a complex on the mRNA (Figure 4). Initially, three initiation factor proteins (IF1, IF2, and IF3) attach to the small ribosomal subunit. This preinitiation complex, along with a methionine-carrying tRNA, then binds to the mRNA near the AUG start codon, forming the initiation complex.

Figure 4: Formation of the Translation Initiation Complex.

This diagram illustrates the assembly of the initiation complex on mRNA. The small ribosomal subunit and an initiator tRNA molecule assemble at the mRNA transcript. The small subunit has three sites: A (amino acid), P (polypeptide), and E (exit). Initiator tRNA carrying methionine binds to the AUG start codon at the ribosome’s P site, becoming the first amino acid in the polypeptide chain. The order of binding differs slightly between prokaryotes and eukaryotes, as detailed in the figure description.

Methionine (Met) is always the first amino acid in new proteins, though it’s not always present in mature proteins, often removed post-translation. Analyzing protein sequences reveals methionine (or formylmethionine) at the N-terminus of all nascent proteins. However, the second amino acid affects methionine retention. For instance, methionine followed by alanine often leads to methionine removal, making alanine the N-terminal amino acid (Table 1). Conversely, methionine followed by lysine typically retains methionine. Table 1 shows N-terminal sequences in prokaryotic and eukaryotic proteins, based on a study of 170 prokaryotic and 120 eukaryotic proteins (Flinta et al., 1986).

Table 1: N-Terminal Sequences of Proteins

N-Terminal Sequence	Percent of Prokaryotic Proteins with This Sequence	Percent of Eukaryotic Proteins with This Sequence
MA*	28.24%	19.17%
MK**	10.59%	2.50%
MS*	9.41%	11.67%
MT*	7.65%	6.67%

* Methionine was removed in all of these proteins
** Methionine was not removed from any of these proteins

Once the initiation complex forms, the large ribosomal subunit joins, releasing initiation factors (IFs). The large subunit contains three tRNA binding sites: the A (amino acid) site where aminoacyl-tRNA anticodons pair with mRNA codons ensuring correct amino acid addition; the P (polypeptide) site where amino acids transfer from tRNA to the growing chain; and the E (exit) site for “empty” tRNA release. Initiator methionine tRNA uniquely binds to the P site, with the A site aligning with the second mRNA codon. The ribosome is now ready for the second aminoacyl-tRNA at the A site, which will link to initiator methionine via the first peptide bond (Figure 5).

Figure 5: Completed Translation Initiation Complex.

The large ribosomal subunit joins the small subunit, finalizing the initiation complex. The initiator tRNA, carrying methionine, is in the P site. The A site is ready for the next tRNA, corresponding to the subsequent codon.

Elongation Phase: Building the Polypeptide Chain

Figure 6: Stages of Translation Elongation.

This figure illustrates the eight key steps of the elongation phase in translation. It shows how the ribosome moves along the mRNA, facilitating tRNA binding, peptide bond formation, and tRNA release, step-by-step.

The elongation phase follows initiation (Figure 6). The ribosome shifts along the mRNA in the 5′-to-3′ direction, a process called translocation requiring elongation factor G. The tRNA matching the second codon then binds to the A site, assisted by elongation factors (EF-Tu and EF-Ts in E. coli) and GTP for energy. GTP hydrolyzes to GDP upon tRNA-amino acid complex binding at the A site, releasing GDP and EF-Tu, which EF-Ts recycles. Next, peptidyl transferase activity forms peptide bonds between adjacent amino acids. rRNA itself, not an enzyme, catalyzes this transferase activity (Pierce, 2000). After peptide bond formation, the ribosome translocates again, moving the tRNA to the E site, from where it’s released to the cytoplasm for amino acid re-attachment. The A site is now vacant, ready for the tRNA of the next codon.

This cycle repeats until all mRNA codons are read, and amino acids are linked in the correct order, forming the polypeptide chain. Translation then needs termination and nascent protein release from the mRNA and ribosome.

Termination of Translation: Releasing the Protein

Termination codons – UAA, UAG, and UGA – signal the end of protein-coding sequences in mRNA. No tRNAs recognize these codons. Instead, release factors bind, prompting mRNA release from the ribosome and ribosome dissociation.

Prokaryotic vs. Eukaryotic Translation: Key Differences

Translation is largely similar in prokaryotes and eukaryotes. While initiation, elongation, and termination factors differ, the genetic code is generally universal. In bacteria, transcription and translation are simultaneous, with short-lived mRNAs. Eukaryotic mRNAs, however, have varied half-lives, undergo modifications, and must exit the nucleus for translation. These extra steps in eukaryotes offer more regulation points for protein production levels, enabling fine-tuning of gene expression.

References and Further Reading

Chapeville, F., et al. On the role of soluble ribonucleic acid in coding for amino acids. Proceedings of the National Academy of Sciences 48, 1086–1092 (1962)

Crick, F. On protein synthesis. Symposia of the Society for Experimental Biology 12, 138–163 (1958)

Flinta, C., et al. Sequence determinants of N-terminal protein processing. European Journal of Biochemistry 154, 193–196 (1986)

Grunberger, D., et al. Codon recognition by enzymatically mischarged valine transfer ribonucleic acid. Science 166, 1635–1637 (1969) doi:10.1126/science.166.3913.1635

Kozak, M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature 308, 241–246 (1984) doi:10.1038308241a0

—. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292 (1986)

—. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research 15, 8125–8148 (1987)

Pierce, B. A. Genetics: A conceptual approach (New York, Freeman, 2000)

Shine, J., & Dalgarno, L. Determinant of cistron specificity in bacterial ribosomes. Nature 254, 34–38 (1975) doi:10.1038/254034a0