Chapter: The Genetic Code - Transcription, RNA Processing, and Translation

Introduction to Gene Expression

DNA stores the hereditary information of life, but DNA itself does not directly perform most cellular functions. DNA is more like an information archive than a working machine. The molecules that carry out most of the work of the cell are proteins. Proteins build cellular structures, speed up chemical reactions, transport materials, receive signals, regulate genes, and help determine how cells function. But before a protein can be made, the information stored in DNA must be used. This process is known as gene expression, the process by which information stored in DNA is used to produce a functional product, usually a protein. Gene expression occurs in two major stages. First, the information in DNA is copied into RNA in a process known as transcription. Second, the information in messenger RNA is used to assemble a chain of amino acids in a process known as translation. The amino acid chain then folds into a protein. This relationship between DNA, RNA, and protein is known as the central dogma of molecular biology: DNA → RNA → Protein.

DNA information flows to RNA and then to a folded protein that performs cellular functions. — **Figure 1. Introduction to Gene Expression.** Gene expression is the process by which information stored in DNA is used to produce a functional product, usually a protein. In protein-coding genes, this involves transcription from DNA to RNA followed by translation from RNA to protein.

The Central Dogma of Molecular Biology

Francis Crick, one of the scientists credited with helping describe the structure of DNA, proposed that DNA functions as an informational storage molecule capable of replicating itself. But he also understood that DNA was not the final working product of most genes. Instead, the information stored in DNA had to be copied and then read by the cell in order to build proteins. According to the central dogma, DNA serves as the template for the synthesis of messenger RNA, or mRNA, during transcription. The mRNA then travels to a ribosome, where the sequence of nucleotides in the mRNA is read and used to assemble a specific sequence of amino acids. Transfer RNA, or tRNA, helps match the nucleotide sequence of the mRNA to the correct amino acids. The amino acids are then joined together by peptide bonds to form a polypeptide chain, which can fold into a functional protein. In this way, DNA stores the instructions, RNA carries and helps interpret the instructions, and proteins carry out much of the work of the cell.

DNA is transcribed into mRNA, which is translated by a ribosome into a polypeptide that folds into a protein. — **Figure 2. The Central Dogma of Molecular Biology.** The central dogma describes the flow of genetic information from DNA to RNA to protein. DNA stores information, mRNA carries the instructions, and ribosomes use those instructions to build proteins.

Exceptions to the Central Dogma

The central dogma describes the most important flow of information in cells, but it is a simplified model. There are important exceptions. Messenger RNA is only one type of RNA. Other RNA molecules, including transfer RNA and ribosomal RNA, are also coded by DNA, but they are not translated into proteins. In these cases, the information flow is simply DNA to RNA. The RNA molecule itself is the final functional product. Another exception involves reverse transcription. Some viruses, such as retroviruses, have RNA genomes. When these viruses infect a host cell, they use an enzyme called reverse transcriptase to synthesize DNA from RNA. In this case, information flows from RNA to DNA, which is the reverse of the usual DNA-to-RNA pathway. Even with these exceptions, the central dogma remains one of the most important ideas in biology. For most protein-coding genes in cells, information flows from DNA to RNA to protein.

The standard DNA to RNA to protein pathway is shown with exceptions for functional RNA and RNA to DNA reverse transcription. — **Figure 3. Exceptions to the Central Dogma.** The central dogma summarizes the major flow of information from DNA to RNA to protein, but some genes produce functional RNAs that are not translated. Reverse transcription also allows information to flow from RNA back to DNA in some viruses.

Structure of DNA and RNA

DNA and RNA are both nucleic acids, but they differ in several important ways. DNA is usually double-stranded, while RNA is usually single-stranded. DNA contains the sugar deoxyribose, while RNA contains the sugar ribose. The difference between these sugars is small, but important. Ribose contains an additional oxygen atom on the 2′ carbon of the sugar molecule. DNA and RNA also differ in one of their nitrogenous bases. DNA uses the bases adenine, thymine, cytosine, and guanine. RNA uses adenine, uracil, cytosine, and guanine. In other words, RNA uses uracil where DNA uses thymine. The monomers of DNA are deoxyribonucleotides. The monomers of RNA are ribonucleotides. In both DNA and RNA, nucleotides are joined together by phosphodiester bonds, producing a strand with directionality. New nucleotides are added to the 3′ end of a growing strand, so nucleic acids are synthesized in the 5′ to 3′ direction.

DNA and RNA are compared by strand structure, sugar type, bases, and 5 prime to 3 prime directionality. — **Figure 4. Structure of DNA and RNA.** DNA and RNA are both nucleic acids, but DNA is usually double-stranded and uses deoxyribose and thymine, while RNA is usually single-stranded and uses ribose and uracil.

Types of RNA

Cells contain several major forms of RNA, each with a specialized role in gene expression or cellular function. Messenger RNA carries genetic instructions copied from DNA to ribosomes. In protein-coding genes, the mature mRNA contains codons that specify the amino acid sequence of a protein. Transfer RNA delivers amino acids to the ribosome during translation. Each tRNA carries a specific amino acid and contains an anticodon that pairs with a complementary codon on the mRNA. This pairing ensures that amino acids are added in the correct order. Ribosomal RNA, or rRNA, combines with proteins to form ribosomes. Ribosomes are the cellular structures that carry out translation. Ribosomal RNA is not merely structural. It also helps catalyze peptide bond formation. Other forms of RNA also exist. Some help regulate gene expression, process other RNA molecules, or defend cells against viruses. RNA is not just a temporary copy of DNA. RNA is an active and versatile molecule in the cell.

mRNA carries codons, tRNA pairs anticodons with codons, and rRNA helps form the ribosome. — **Figure 5. Types of RNA.** Cells use several forms of RNA. Messenger RNA carries codons, transfer RNA matches codons with amino acids, and ribosomal RNA helps form ribosomes and catalyze peptide bond formation.

The Triplet Code

Once biologists understood the central dogma, they understood the general pattern of information flow in the cell. But that raised a new question. How does the sequence of nucleotides in a strand of mRNA code for the sequence of amino acids in a protein? Proteins are built from 20 common amino acids. RNA, however, is made from only four types of nucleotides: adenine, uracil, cytosine, and guanine. If one nucleotide coded for one amino acid, there would only be four possible code words. That would not be enough to code for 20 amino acids. If two nucleotides coded for one amino acid, there would be 4 × 4, or 16, possible code words. That still would not be enough. A three-nucleotide code produces 4 × 4 × 4, or 64, possible code words. This is more than enough to code for 20 amino acids. This is why the genetic code is a triplet code. Each three-nucleotide sequence in mRNA is called a codon. Each codon specifies either a particular amino acid or a translation signal, such as start or stop.

One-, two-, and three-nucleotide codes are compared, showing that triplet codons produce 64 possible code words. — **Figure 6. The Triplet Code.** The genetic code is a triplet code because three RNA nucleotides form one codon. A three-nucleotide code creates 64 possible codons, enough to specify 20 amino acids and translation signals.

Properties of the Genetic Code

The genetic code has several important properties. First, the genetic code is a triplet code because each codon consists of three RNA nucleotides. Second, the genetic code is redundant because there are 64 possible codons but only 20 common amino acids. Therefore, many amino acids are specified by more than one codon. For example, CCU and CCC both code for the amino acid proline. This redundancy helps reduce the effects of some mutations because a change in one nucleotide does not always change the amino acid. Third, the genetic code is unambiguous because a specific codon specifies only one amino acid or one translation signal. For example, UUU codes for phenylalanine and does not code for any other amino acid. Fourth, the genetic code is nearly universal. With only a few exceptions, organisms use the same codons to specify the same amino acids. Bacteria, archaea, plants, animals, fungi, and protists all use essentially the same genetic code. This strongly suggests that the genetic code evolved early in the history of life and was inherited from a common ancestor. Finally, the genetic code is conservative. In many cases, codons that differ only in the third nucleotide code for the same amino acid or for chemically similar amino acids. This is part of the reason that mutations in the third position of a codon are often less disruptive than mutations in the first or second position.

Panels show the genetic code as triplet, redundant, unambiguous, nearly universal, and conservative. — **Figure 7. Properties of the Genetic Code.** The genetic code is triplet, redundant, unambiguous, nearly universal, and conservative. These properties help explain how codons specify amino acids and why some mutations have smaller effects than others.

Start and Stop Codons

The start codon marks the beginning of translation. The most common start codon is AUG. AUG codes for methionine in eukaryotes and a modified form of methionine in many bacteria. Although AUG is the standard start codon, some organisms and genes can use alternative start codons. Still, AUG is by far the most common starting signal used in translation. Stop codons mark the end of translation. Stop codons do not code for amino acids. Instead, they signal the ribosome to stop building the polypeptide chain. The three stop codons are UAA, UAG, and UGA.

An mRNA strand shows AUG starting translation and UAA, UAG, and UGA acting as stop codons. — **Figure 8. Start and Stop Codons.** AUG is the most common start codon and usually marks where translation begins. Stop codons do not code for amino acids; they signal the ribosome to stop translation.

Predicting Proteins from DNA

Once the genetic code was deciphered, scientists could use a DNA sequence to predict the amino acid sequence of a protein. For a protein-coding gene, only one DNA strand is used as the template strand during transcription. The other strand is called the coding strand because its sequence is nearly identical to the mRNA sequence, except that DNA contains thymine and RNA contains uracil. For example, consider the DNA template strand 3′ - TAC GTC TAG TCC ATC - 5′. During transcription, RNA nucleotides pair with the template strand according to complementary base-pairing rules: DNA A pairs with RNA U, DNA T pairs with RNA A, DNA C pairs with RNA G, and DNA G pairs with RNA C. This produces the mRNA sequence 5′ - AUG CAG AUC AGG UAG - 3′. The mRNA can then be translated using the genetic code. AUG codes for methionine and usually serves as the start codon, CAG codes for glutamine, AUC codes for isoleucine, AGG codes for arginine, and UAG is a stop codon. Therefore, this sequence would produce the amino acid chain methionine - glutamine - isoleucine - arginine. Translation ends when the ribosome reaches the stop codon.

A DNA template is transcribed into mRNA codons, which translate into methionine, glutamine, isoleucine, and arginine. — **Figure 9. Predicting Proteins from DNA.** A DNA template strand is transcribed into complementary mRNA, and the mRNA codons are translated into a specific amino acid sequence. Translation stops when the ribosome reaches a stop codon.

Using an Amino Acid Chart

Once you have transcribed a DNA template strand into mRNA, you can use an amino acid chart to predict the amino acid sequence of the protein. The most important thing to remember is that the chart is read using mRNA codons, not DNA triplets. First, identify the DNA template strand and transcribe it into mRNA using complementary base-pairing rules: A pairs with U, T pairs with A, C pairs with G, and G pairs with C. Be sure to write the mRNA in the 5′ to 3′ direction. Next, divide the mRNA sequence into groups of three nucleotides, called codons. Then use the amino acid chart to determine what each codon specifies. Most charts are read by finding the first base of the codon on the left side, the second base across the top, and the third base on the right side or within the box. For example, if the DNA template strand is 3′ - TAC GTC TAG TCC ATC - 5′, the mRNA sequence is 5′ - AUG CAG AUC AGG UAG - 3′. Breaking that into codons gives AUG | CAG | AUC | AGG | UAG. Using the chart, AUG codes for methionine, CAG codes for glutamine, AUC codes for isoleucine, and AGG codes for arginine. UAG is a stop codon, so translation ends there. Therefore, the predicted amino acid sequence is methionine - glutamine - isoleucine - arginine. As a general rule, begin reading at the start codon, usually AUG, and continue codon by codon until you reach a stop codon, which signals the end of the protein.

All 64 codons are shown. AUG is highlighted as the start codon for methionine, and UAA, UAG, and UGA are marked as stop codons. A small callout shows example translations: AUG to methionine, CAG to glutamine, and UAG to stop. — **Figure 10. The Genetic Code: Amino Acid Chart.** The standard genetic code chart shows how mRNA codons are translated into amino acids using the RNA bases U, C, A, and G. Students read the first base, second base, and third base of each codon to determine the correct amino acid. The chart includes all 64 codons, highlights AUG as the start codon coding for methionine, and labels UAA, UAG, and UGA as stop codons. A side example demonstrates how codons such as AUG → methionine, CAG → glutamine, and UAG → stop are identified using the chart.

Predicting Amino Acids from a DNA Template Strand

Transcribe the DNA template strand into mRNA, divide the mRNA into codons, and use the genetic code to identify the first three amino acids in the predicted protein sequence.

Instructions

The strand below is a DNA template strand, written 3′ to 5′. First, transcribe the template strand into mRNA written 5′ to 3′. Then divide the mRNA into three-base codons. Finally, use the genetic code chart to identify the first three amino acids.

DNA template strand: 3′ - TAC GTC TAG TCC ATC - 5′

Amino Acid Chart

This chart uses mRNA codons. Read each codon by identifying its first base, second base, and third base. Do not use DNA triplets in the chart.

Standard Genetic Code for mRNA Codons
First base	Second base U	Second base C	Second base A	Second base G
U	UUUPhenylalanine UUCPhenylalanine UUALeucine UUGLeucine	UCUSerine UCCSerine UCASerine UCGSerine	UAUTyrosine UACTyrosine UAAStop UAGStop	UGUCysteine UGCCysteine UGAStop UGGTryptophan
C	CUULeucine CUCLeucine CUALeucine CUGLeucine	CCUProline CCCProline CCAProline CCGProline	CAUHistidine CACHistidine CAAGlutamine CAGGlutamine	CGUArginine CGCArginine CGAArginine CGGArginine
A	AUUIsoleucine AUCIsoleucine AUAIsoleucine AUGMethionine / Start	ACUThreonine ACCThreonine ACAThreonine ACGThreonine	AAUAsparagine AACAsparagine AAALysine AAGLysine	AGUSerine AGCSerine AGAArginine AGGArginine
G	GUUValine GUCValine GUAValine GUGValine	GCUAlanine GCCAlanine GCAAlanine GCGAlanine	GAUAspartic acid GACAspartic acid GAAGlutamic acid GAGGlutamic acid	GGUGlycine GGCGlycine GGAGlycine GGGGlycine

Example: the mRNA codon AUG is read as first base A, second base U, third base G. In the chart, AUG codes for methionine and usually serves as the start codon. The codons UAA, UAG, and UGA are stop codons.

Quiz: Identify the First Three Amino Acids

Select the amino acid coded by each of the first three mRNA codons. Remember: the amino acid chart is read using mRNA codons, not DNA triplets.

Amino Acid 1

Choose the amino acid coded by the first mRNA codon.

Amino Acid 2

Choose the amino acid coded by the second mRNA codon.

Amino Acid 3

Choose the amino acid coded by the third mRNA codon.

How to Work Through the Sequence

The amino acid chart is read using mRNA codons. The DNA template strand must be transcribed into mRNA first.

DNA template: 3′ - TAC GTC TAG TCC ATC - 5′
mRNA: 5′ - AUG CAG AUC AGG UAG - 3′
Codons: AUG | CAG | AUC | AGG | UAG
First three amino acids: methionine, glutamine, isoleucine

AUG → methionine CAG → glutamine AUC → isoleucine AGG → arginine UAG → stop

Mutations

A mutation is a permanent change in an organism’s DNA sequence. Mutations are important because they create new alleles, or new versions of genes. These new alleles may produce different versions of proteins, which can alter cellular function and influence an organism’s phenotype. For this reason, mutations are one of the ultimate sources of biological diversity. Without mutations, there would be no new genetic variation for natural selection, genetic drift, or other evolutionary processes to act on. Mutations can also affect an organism’s fitness, or its ability to survive and reproduce. A mutation that increases fitness is considered beneficial, while a mutation that decreases fitness is deleterious. A mutation that has no meaningful effect on fitness is neutral. Mutations can be grouped into two broad categories: point mutations and chromosome-level mutations.

A DNA mutation creates a new allele that may affect protein function, phenotype, and fitness. — **Figure 11. Mutations.** A mutation is a permanent change in DNA sequence. Mutations create new alleles that may affect proteins, phenotypes, and fitness, making them an ultimate source of genetic variation.

Mutations: Point Mutations

Point mutations involve changes to one nucleotide. They often occur when DNA is copied during DNA replication and the cell’s proofreading mechanisms fail to correct an error. A substitution occurs when one nucleotide is replaced by another. Because the genetic code is redundant, a substitution does not always change the amino acid sequence. A silent mutation occurs when a nucleotide change does not change the amino acid. For example, if an mRNA codon changes from AUU to AUA, both codons still code for isoleucine. Silent mutations are especially common when the third nucleotide of a codon changes, a result of the conservative property of the code. A missense mutation occurs when a nucleotide change causes one amino acid to be replaced by another. This can affect the structure or function of the protein. Some missense mutations have little effect, while others can strongly affect phenotype. A single point mutation can sometimes produce a visible change in phenotype. For example, in some mice, a single nucleotide change alters one amino acid in a protein involved in pigment production. In one version of the protein, the amino acid arginine is present at a specific position; in the mutated version, cysteine occurs in that same position. That one amino acid change can alter the function of the protein enough to change fur color. If one fur color provides better camouflage in a dark environment, while another provides better camouflage in a light environment, mice with different phenotypes may become more successful in different habitats. Over time, this ecological separation can reduce mating between the groups. If the groups continue to experience different selection pressures and exchange fewer genes, they may begin to diverge genetically. This is one way a simple mutation can contribute to the early stages of genetic isolation and, eventually, speciation.

A single nucleotide substitution changes an mRNA codon, producing either a silent or missense mutation. — **Figure 12. Point Mutations.** Point mutations change a single nucleotide. Some substitutions are silent, while others change an amino acid and may alter protein function, phenotype, and fitness.

Mutations: Chromosome-Level Mutations

Chromosome-level mutations involve large changes to DNA. These may include the loss, duplication, inversion, or movement of chromosome segments. They may also involve changes in the number of entire chromosomes. A deletion occurs when part of a chromosome is lost. A duplication occurs when part of a chromosome is copied one or more extra times. An inversion occurs when a chromosome segment breaks, flips, and reattaches in the reverse orientation. Inversions may disrupt genes, alter gene regulation, or interfere with normal chromosome pairing during meiosis. A translocation occurs when a segment of one chromosome breaks off and attaches to a different chromosome. Translocations can disrupt genes or create new gene combinations. Mistakes can also occur during cell division. During meiosis, homologous chromosomes or sister chromatids are supposed to separate into different gametes. If chromosomes fail to separate properly, the result is called nondisjunction. After fertilization, the resulting organism may have too many or too few copies of a chromosome. In humans, Down syndrome is usually caused by trisomy 21, meaning an individual has three copies of chromosome 21 instead of two. Some organisms can also have extra complete sets of chromosomes. This condition is known as polyploidy. Polyploidy is rare in animals but relatively common in plants. In some cases, polyploidy can contribute to the formation of new plant species because polyploid plants may become reproductively isolated from diploid plants. Most humans have 23 pairs of homologous chromosomes, for a total of 46 chromosomes. One chromosome in each pair comes from the mother and one comes from the father. Homologous chromosomes are similar in size and carry the same sequence of genes, but they may carry different alleles of those genes.

Transcription: Overview

Transcription is the process by which RNA is synthesized using DNA as a template. During transcription, one strand of DNA serves as the template strand. RNA nucleotides pair with exposed DNA nucleotides according to complementary base-pairing rules. The opposite DNA strand is known as the coding strand. The coding strand has nearly the same sequence as the RNA transcript, except that the coding strand contains thymine and the RNA transcript contains uracil. Unlike DNA replication, transcription usually copies only a specific section of DNA. This section is a gene. A gene is a segment of DNA that contains the instructions for producing a functional product, such as a protein or functional RNA molecule. During transcription, RNA is synthesized in the 5′ to 3′ direction. This means that ribonucleotides are added to the 3′ end of the growing RNA strand. The ribonucleotides are joined together by phosphodiester bonds.

RNA is synthesized from one DNA template strand, with the transcript growing in the 5 prime to 3 prime direction. — **Figure 14. Transcription.** Transcription synthesizes RNA from a DNA template strand. RNA nucleotides are added to the 3′ end of the growing RNA transcript, so RNA is synthesized in the 5′ to 3′ direction.

Transcription: RNA Polymerase

The enzyme responsible for RNA synthesis is RNA polymerase. RNA polymerase reads the DNA template strand and builds a complementary RNA molecule. RNA polymerase is similar to DNA polymerase in that it builds a nucleic acid strand using complementary base pairing. However, RNA polymerase differs from DNA polymerase in one important way: RNA polymerase does not require a primer. It can begin RNA synthesis directly. RNA polymerase also temporarily opens the DNA double helix during transcription. As it moves along the DNA template, it unwinds a short region of DNA, adds ribonucleotides to the growing RNA transcript, and allows the DNA strands behind it to re-form the double helix. Bacteria possess one major type of RNA polymerase that can synthesize different forms of RNA. Eukaryotic cells possess three primary RNA polymerases. RNA polymerase I synthesizes most ribosomal RNA. RNA polymerase II synthesizes messenger RNA and some regulatory RNAs. RNA polymerase III synthesizes transfer RNA and some other small RNAs.

RNA polymerase opens DNA, reads the template strand, and builds RNA without needing a primer. — **Figure 15. RNA Polymerase.** RNA polymerase reads a DNA template strand and builds a complementary RNA molecule. Unlike DNA polymerase, RNA polymerase can begin RNA synthesis without a primer.

Transcription: Initiation of Transcription in Prokaryotes

Transcription begins at specific DNA sequences known as promoters. A promoter marks where transcription should begin. Genes are not active at all times. Cells turn genes on and off depending on the needs of the cell and the conditions of the environment. When a gene is activated, RNA polymerase must be guided to the correct promoter region. In bacteria, transcription is initiated by a protein known as a sigma factor. The sigma factor recognizes and binds to a promoter sequence. It then helps guide RNA polymerase to the correct starting point on the DNA. Different sigma factors recognize different promoter sequences. This allows bacterial cells to activate different groups of genes under different environmental conditions. For example, one set of genes may be activated during heat stress, while another set may be activated when a specific nutrient becomes available. Once the sigma factor binds to the promoter, RNA polymerase attaches to the sigma factor-DNA complex. RNA polymerase locally unwinds the DNA double helix, exposing the template strand. Ribonucleotides enter the active site of RNA polymerase and pair with complementary DNA bases. Once several ribonucleotides have been joined together, the sigma factor detaches. The sigma factor can then be reused to initiate transcription at another promoter. At this point, transcription enters the elongation phase.

A sigma factor binds a bacterial promoter and guides RNA polymerase to start transcription. — **Figure 16. Prokaryotic Transcription Initiation.** In bacteria, sigma factors recognize promoter sequences and guide RNA polymerase to the correct starting point. After transcription begins, the sigma factor detaches and can be reused.

Transcription: Elongation of Transcription

During elongation, RNA polymerase moves along the DNA template strand and synthesizes RNA. As RNA polymerase travels down the DNA molecule, it continuously unwinds a short section of DNA ahead of itself. Incoming ribonucleotides pair with exposed DNA bases according to complementary base-pairing rules. RNA polymerase catalyzes the formation of phosphodiester bonds between adjacent ribonucleotides. This causes the RNA transcript to elongate at its 3′ end. Behind RNA polymerase, the DNA strands reassociate and reform the double helix. The growing RNA strand exits RNA polymerase as synthesis continues. The DNA molecule is not permanently altered by transcription. It is used as a template, not consumed.

**Figure 17. Transcription Elongation.** During elongation, RNA polymerase moves along the DNA template strand and adds ribonucleotides to the growing RNA transcript. The DNA behind the enzyme re-forms its double helix.

Transcription: Termination of Transcription in Prokaryotes

Transcription ends when RNA polymerase encounters a termination signal. In bacteria, one common termination mechanism involves the formation of an RNA hairpin. As a specific RNA sequence is synthesized, complementary bases within the RNA molecule pair with each other. This causes the RNA to fold back on itself, forming a stem-loop structure known as a hairpin. The hairpin destabilizes the interaction between the RNA transcript and the DNA template. As a result, the RNA transcript is released, RNA polymerase detaches from the DNA, and the DNA double helix fully reforms.

**Figure 18. Prokaryotic Transcription Termination.** In many bacterial genes, transcription can end when the RNA transcript folds into a hairpin. This structure destabilizes the transcription complex, releasing the RNA and RNA polymerase.

Transcription: Transcription in Eukaryotes

The overall mechanism of transcription in eukaryotes is similar to transcription in prokaryotes, but there are several important differences. First, eukaryotic DNA is enclosed within a nucleus. This means transcription occurs in the nucleus, while translation occurs later in the cytoplasm. Second, eukaryotes use multiple RNA polymerases. RNA polymerase I synthesizes most ribosomal RNA. RNA polymerase II synthesizes messenger RNA from protein-coding genes. RNA polymerase III synthesizes transfer RNA and some additional small RNA molecules. Third, eukaryotic transcription requires numerous proteins known as transcription factors. These transcription factors assemble at the promoter region along with RNA polymerase II to form a transcription initiation complex. One important promoter sequence found in many eukaryotic genes is the TATA box. Once the transcription initiation complex forms, RNA polymerase II unwinds the DNA and begins RNA synthesis. Elongation in eukaryotes proceeds in much the same way as bacterial transcription. However, termination differs. In eukaryotes, proteins cleave the newly synthesized RNA transcript, and RNA polymerase eventually detaches from the DNA template. Unlike bacterial mRNA, eukaryotic RNA transcripts usually require extensive processing before they become functional.

Transcription factors and RNA polymerase II assemble at a eukaryotic promoter inside the nucleus. — **Figure 19. Eukaryotic Transcription.** Eukaryotic transcription occurs in the nucleus and requires transcription factors to help assemble RNA polymerase II at the promoter. The initial RNA product is usually processed before it becomes functional mRNA.

Transcription: The Primary Transcript

The initial RNA molecule synthesized by RNA polymerase II is known as the primary transcript, or pre-mRNA. The primary transcript contains both coding and noncoding regions. The coding regions are known as exons because they remain in the mature mRNA that exits the nucleus. The noncoding regions are known as introns. Before the RNA can function as mRNA, the introns must be removed and the exons must be joined together. This process is known as RNA processing or post-transcriptional modification. After processing, the molecule is known as mature mRNA.

A primary transcript contains exons and introns before processing creates mature mRNA. — **Figure 20. The Primary Transcript.** The primary transcript, or pre-mRNA, is the initial RNA molecule made by RNA polymerase II. It contains exons that remain in mature mRNA and introns that are removed during RNA processing.

RNA Processing in Eukaryotes

In bacteria, transcription from DNA to mRNA can be a direct pathway. In eukaryotes, however, the primary transcript must be modified before it leaves the nucleus. The eukaryotic primary transcript undergoes three major modifications: addition of a 5′ cap, addition of a poly-A tail, and RNA splicing. Soon after transcription begins, a modified guanine nucleotide is attached to the 5′ end of the primary transcript. This structure is called the 5′ cap. The 5′ cap helps protect the RNA from degradation and helps ribosomes recognize the mRNA during translation. At the 3′ end of the pre-mRNA, a long series of adenine nucleotides is added. This structure is called the poly-A tail. The poly-A tail increases RNA stability and assists with export of the mRNA from the nucleus. The third major modification is RNA splicing. During RNA splicing, introns are removed from the pre-mRNA and exons are joined together. The resulting mature mRNA contains the sequence that will be translated into a protein. RNA splicing is carried out by a molecular complex known as the spliceosome. The spliceosome is made of proteins and small nuclear RNAs organized into structures called small nuclear ribonucleoproteins, or snRNPs. Specific snRNPs recognize nucleotide sequences that mark the boundaries between exons and introns. The spliceosome then cuts the RNA at both ends of the intron, removes the intron as a looped lariat structure, and joins the adjacent exons together. The removed intron is broken down, and its ribonucleotides can be recycled by the cell. The mature mRNA is then ready to leave the nucleus.

Pre-mRNA receives a 5 prime cap, poly-A tail, and splicing to remove introns and join exons. — **Figure 21. RNA Processing in Eukaryotes.** Eukaryotic pre-mRNA is modified by addition of a 5′ cap, addition of a poly-A tail, and RNA splicing. Splicing removes introns and joins exons to produce mature mRNA.

Export of mRNA from the Nucleus

Once RNA processing is complete, mature mRNA exits the nucleus through structures known as nuclear pores. The mRNA then enters the cytoplasm, where it can attach to a ribosome and undergo translation. This separation of transcription and translation is one of the major differences between eukaryotic and prokaryotic gene expression. In bacteria, transcription and translation can occur simultaneously because bacteria lack a nucleus. In eukaryotes, transcription and translation are physically separated because transcription occurs in the nucleus and translation occurs in the cytoplasm.

Processed mRNA exits the nucleus through a nuclear pore and moves to the cytoplasm for translation. — **Figure 22. Export of mRNA from the Nucleus.** After eukaryotic mRNA is processed, it exits the nucleus through nuclear pores. The mature mRNA enters the cytoplasm, where it can attach to a ribosome and be translated.

Translation: Overview

Translation is the process by which the nucleotide sequence of mRNA is converted into the amino acid sequence of a protein. During translation, the ribosome reads the mRNA in groups of three nucleotides, known as codons. Each codon specifies a particular amino acid or a translation signal. The sequence of codons in the mRNA determines the sequence of amino acids in the protein. Translation requires three major components: mRNA, tRNA, and ribosomes. Messenger RNA carries the codon sequence, transfer RNA brings amino acids to the ribosome, and ribosomes hold the mRNA and tRNAs in position and catalyze peptide bond formation.

A ribosome reads mRNA codons while tRNAs bring amino acids to build a growing polypeptide. — **Figure 23. Translation.** Translation converts the nucleotide sequence of mRNA into the amino acid sequence of a protein. Ribosomes read codons, tRNAs bring amino acids, and peptide bonds join amino acids into a polypeptide.

Translation: Transfer RNA

Transfer RNA, or tRNA, serves as the molecular bridge between mRNA codons and amino acids. Each tRNA molecule is attached to a specific amino acid. At the opposite end of the tRNA is a three-nucleotide sequence known as the anticodon. The anticodon pairs with a complementary codon on the mRNA. For example, the mRNA codon AUG pairs with the tRNA anticodon UAC. The tRNA with this anticodon carries methionine. Specialized enzymes attach the correct amino acid to each tRNA molecule. This process is known as charging. Charging is essential because the ribosome does not independently verify whether the correct amino acid is attached to the tRNA. If the wrong amino acid is attached, the ribosome may still add it to the growing polypeptide chain.

A tRNA carries an amino acid and pairs its anticodon with a complementary mRNA codon. — **Figure 24. Transfer RNA.** Transfer RNA connects mRNA codons to amino acids. Each tRNA carries a specific amino acid and uses its anticodon to pair with a complementary codon on the mRNA.

Translation: Ribosomes

Ribosomes are the molecular machines responsible for protein synthesis. They are made of ribosomal RNA and proteins organized into two subunits: a small subunit and a large subunit. The small subunit binds the mRNA and helps position it correctly. The large subunit catalyzes peptide bond formation between amino acids. Ribosomes contain three major tRNA binding sites. The A site, or aminoacyl site, receives the incoming tRNA carrying the next amino acid. The P site, or peptidyl site, holds the tRNA carrying the growing polypeptide chain. The E site, or exit site, releases the empty tRNA from the ribosome. A simple way to remember the order is A-P-E: arrival, peptide bond formation, and exit.

A ribosome shows small and large subunits with A, P, and E sites for tRNA movement during translation. — **Figure 25. Ribosomes.** Ribosomes coordinate translation using small and large subunits. Their A, P, and E sites position tRNAs for arrival, peptide bond formation, and exit.

Translation: Initiation of Translation

Translation begins when the small ribosomal subunit binds to the mRNA and positions itself at the start codon. In bacteria, the ribosome recognizes a ribosome-binding site. This sequence helps position the ribosome at the correct starting location on the mRNA. In eukaryotes, the small ribosomal subunit typically binds near the 5′ cap and scans along the mRNA until it reaches the start codon. The start codon is usually AUG. A specialized initiator tRNA with the anticodon UAC, carrying methionine binds to the start codon. In many bacteria, the first amino acid is a modified form of methionine called formylmethionine, or fMet. In eukaryotes, the first amino acid is usually methionine. Once the initiator tRNA is positioned correctly, the large ribosomal subunit attaches. The initiator tRNA begins in the P site, which positions the ribosome for elongation.

The ribosome assembles at AUG with an initiator tRNA in the P site to begin translation. — **Figure 26. Translation Initiation.** Translation begins when the ribosome is positioned at the start codon, usually AUG. The initiator tRNA pairs with the start codon, and the large ribosomal subunit joins to begin elongation.

Translation: Elongation of Translation

During elongation, amino acids are added one by one to the growing polypeptide chain. First, a charged tRNA enters the A site. Its anticodon pairs with the complementary codon on the mRNA. Next, the ribosome catalyzes the formation of a peptide bond between the amino acid in the P site and the amino acid in the A site. This transfers the growing polypeptide chain onto the tRNA in the A site. Then the ribosome shifts forward by one codon. This movement is called translocation. As the ribosome moves, the tRNA in the A site shifts to the P site. The empty tRNA in the P site shifts to the E site and exits the ribosome. A new codon is now exposed in the A site. This cycle repeats as the ribosome reads each codon and adds the correct amino acid to the growing polypeptide chain.

A ribosome elongates a polypeptide as tRNAs move through the A, P, and E sites. — **Figure 27. Translation Elongation.** During elongation, charged tRNAs enter the A site, peptide bonds form, and the ribosome translocates along the mRNA. This cycle adds amino acids one by one to the growing polypeptide.

Translation: Termination of Translation

Translation ends when the ribosome encounters a stop codon. The three stop codons are UAA, UAG, and UGA. Stop codons do not code for amino acids and are not recognized by tRNAs carrying amino acids. Instead, proteins known as release factors bind to the stop codon in the A site. The release factor causes the completed polypeptide chain to be released from the ribosome. The ribosomal subunits then separate from the mRNA and can be reused in additional rounds of translation.

Alt text: A release factor binds a stop codon, releasing the completed polypeptide and separating the ribosome. — **Figure 28. Translation Termination.** Translation ends when the ribosome reaches a stop codon. A release factor binds, the completed polypeptide is released, and the ribosomal subunits separate for reuse.

Coupled Transcription and Translation in Bacteria

Because bacteria lack a nucleus, transcription and translation can occur simultaneously. As RNA polymerase synthesizes mRNA, ribosomes can attach to the emerging transcript and begin translation before transcription is complete. This process is called coupled transcription and translation. It allows bacterial cells to respond quickly to environmental changes. If a bacterium needs a particular protein, it can begin producing that protein almost immediately after transcription begins.

In a bacterial cell, ribosomes translate an mRNA while RNA polymerase is still transcribing it. — **Figure 29. Coupled Transcription and Translation in Bacteria.** Because bacteria lack a nucleus, transcription and translation can occur at the same time. Ribosomes can begin translating an mRNA while RNA polymerase is still synthesizing it.

Decoupled Transcription and Translation in Eukaryotes

In eukaryotic cells, transcription and translation are separated into different cellular compartments. Transcription occurs inside the nucleus, where RNA polymerase synthesizes a primary transcript from DNA. This RNA molecule is processed by the addition of a 5′ cap, addition of a poly-A tail, and removal of introns through RNA splicing. The mature mRNA then exits the nucleus through nuclear pores. Translation occurs in the cytoplasm. Some ribosomes are free in the cytoplasm, while others are attached to the rough endoplasmic reticulum. Free ribosomes generally synthesize proteins that function in the cytosol. Ribosomes attached to the rough endoplasmic reticulum often synthesize proteins that will be inserted into membranes, secreted from the cell, or sent to certain organelles. This separation gives eukaryotic cells more control over gene expression. Before an mRNA is translated, it can be processed, regulated, transported, stored, or degraded.

Eukaryotic transcription occurs in the nucleus, while processed mRNA is translated in the cytoplasm or rough ER. — **Figure 30. Decoupled Transcription and Translation in Eukaryotes.** In eukaryotes, transcription occurs in the nucleus and translation occurs in the cytoplasm or on rough ER ribosomes. This separation allows RNA to be processed, regulated, exported, stored, or degraded before translation.

Regulation of Gene Expression

Nearly all cells in a multicellular organism contain the same DNA, but they do not use every gene at the same time. Cells regulate gene expression to control which proteins are produced, when they are produced, and how much is made. This regulation allows cells with the same genome to develop different structures and functions. For example, muscle cells activate genes involved in contraction and energy use, while neurons activate genes needed for electrical signaling and communication with other cells. Gene expression can be regulated at many stages. Tightly packed chromatin makes DNA less accessible for transcription, while loosely packed chromatin allows genes to be transcribed more easily. Transcription factors can increase or decrease transcription by affecting RNA polymerase activity. After transcription, RNA processing and alternative splicing determine which exons remain in the mature mRNA, while mRNA stability affects how long the transcript remains available for translation. Cells can also regulate whether ribosomes translate an mRNA. Even after a protein is produced, it may be folded, modified, activated, transported to a specific location, or degraded when it is no longer needed.