Cover1 - Chromosome Structure2 - Chromosome Compaction3 - Chromosome Variation5 - Nucleic Acid Structure6 - DNA Replication7 - Mutations and DNA Repair8 - Polymerase Chain Reaction (PCR)9 - Transcription10 - RNA Modifications11 - Translation12 - Gene Cloning13 - The lac Operon14 - Gene Regulation in Eukaryotes15 - Epigenetics16 - Genome Editing

9 - Transcription

When a gene is activated, the gene is transcribed, producing an RNA intermediate that can be later converted into a polypeptide by translation. Structural genes are those genes that are transcribed to produce messenger RNA (mRNA) molecules. The mRNA molecule is then translated to make a protein. Nonstructural genes are also transcribed to produce RNA molecules; however, the RNA molecule is not translated and instead functions directly in the cell. These functional RNA molecules are called noncoding RNAs (ncRNAs). Noncoding RNAs include transfer RNA molecules (tRNAs), ribosomal RNA molecules (rRNAs), and the Xist and Tsix RNA molecules discussed in an earlier section.

Key Questions

  • What is the difference between a structural and a nonstructural gene?

A. Transcription in Bacteria

Expression of Structural Genes

What factors determine whether a structural gene is expressed; in other words, the gene is activated to make a mRNA molecule?  Expression requires the interaction between transcription factor proteins and specific DNA sequences near the gene.

The DNA sequences that regulate the expression of a particular gene include the following (see figure 9.1):

Transcription produces an RNA molecule that is complementary to the template or antisense strand of DNA. The other DNA strand, the one that forms hydrogen bonds with the template DNA strand, is called the coding or sense DNA strand. The coding DNA strand is identical in sequence to the RNA transcript, except that the RNA molecule contains uracil (U) instead of thymine (T).

8.1_Transcription_Overview_1.jpg
Figure 9.1 DNA sequences that control transcription.  --- Image created by SL

Key Questions

  • What are the functions of the three DNA sequences that regulate transcription?
  • What is the difference between the template and the coding DNA strands?

Transcription Stages

Transcription in the bacterium E. coli has the following three stages (see figure 9.2):

  1. Initiation. During the initiation stage, a transcription factor protein called sigma (σ) factor guides the RNA polymerase to the promoter.
  2. Elongation. During elongation, the RNA polymerase bound to the promoter separates the two DNA strands, forming an open complex. RNA polymerase then reads the template DNA strand while synthesizing a complementary mRNA transcript.
  3. Termination. The termination stage involves the release of the RNA polymerase and the mRNA transcript from the DNA.
8.2_Transcription_Overview.jpg
Figure 9.2 Transcription Overview --- Image created by SL.

Key Questions

  • What is happening during the three stages of transcription?
  • What is the function of sigma factor?

Promoter Structure in Bacteria

The bacterial promoter is located adjacent or upstream of the gene to be transcribed and serves as a docking site for the sigma (σ) factor protein and later RNA polymerase. Important DNA sequence elements within the promoter are numbered in relation to the +1 site, the first nucleotide in the template DNA strand that is transcribed (see figure 9.3). Important DNA sequence elements within the bacterial promoter include the following:

Both the -35 and -10 sequences (5’-TTGACA-3’ and 5’-TATAAT-3’, respectively) are consensus sequences, meaning that they are the “average” sequences found by comparing the DNA sequences of many E. coli promoters. Some bacterial promoters are strong promoters, whereas others are weak promoters. The difference between strong and weak promoters largely depends on how closely the promoter DNA sequence matches the -35 and -10 consensus sequences.  Strong promoters initiate transcription frequently, while weak promoters initiate transcription less frequently.

8.3_Bacterial_Promoter.jpg
Figure 9.3 Bacterial Promoter --- Image created by SL

Key Questions

  • What is the function of the -35 sequence?
  • What are the two functions of the -10 sequence?
  • What is the function of the +1 site?
  • What is the difference between a strong and a weak promoter?

Bacterial RNA Polymerase

In E. coli, the RNA polymerase core enzyme is composed of five protein subunits (α1, α2, β, β’, and ω) (see figure 9.4). The two α subunits and the ω subunit function to assemble the enzyme and bind to the DNA sequence to be transcribed. The RNA molecule is synthesized between the β and β’ subunits.

The RNA polymerase core enzyme associates with a sigma (σ) factor protein. The σ factor protein is an example transcription factor.  E. coli makes at least eight different types of sigma factor proteins, depending on the environmental conditions encountered by the cell. The main sigma factor in E. coli is called the housekeeping sigma factor or σ70. σ70 functions to guide the RNA polymerase core enzyme to the promoters of genes that are required to maintain the E. coli cell. In addition to σ70, there are specialized sigma factor proteins that guide the RNA polymerase core enzyme to survival genes when an E. coli cell encounters stressful environments. These specialized sigma factors include a nitrogen starvation sigma factor (σ54), a general starvation sigma factor (σ38), and a heat shock sigma factor (σ32).

Sigma factor proteins contain a helix-turn-helix motif (a protein tertiary structure) that binds to the major groove of the DNA within the -35 and -10 sequences within the promoter. We will learn about the helix-turn-helix motif in more detail in Part 14.

Sigma factor and the RNA polymerase core enzyme associate to form the RNA polymerase holoenzyme.

8.4_RNA_Polymerase_Holoenzyme_Subunits.jpg
Figure 9.4 RNA Polymerase Holoenzyme Subunits --- Image created by SL

Key Questions

  • Why does E. coli make several different types of sigma factor proteins?
  • What is the difference between the RNA polymerase core enzyme and the RNA polymerase holoenzyme?

Transcription Initiation in Bacteria

Transcription initiation in bacteria occurs as follows:

  1. The RNA polymerase holoenzyme recognizes the promoter via sigma (σ) factor interaction with the -35 and -10 sequences. At this stage, the RNA polymerase holoenzyme:DNA complex is called a closed complex because the two DNA strands are still hydrogen bonded together.
  2. The hydrogen bonds within the -10 sequence (containing multiple AT base pairs) are broken forming an open complex. The RNA polymerase core enzyme is the helicase that separates the two DNA strands at the -10 sequence.
  3. A short RNA molecule is synthesized beginning at the +1 sequence; however, the RNA polymerase core enzyme is still tethered to σ factor. Sigma (σ) factor is still bound to the -10 and -35 sequences in the DNA.
  4. The sigma (σ) factor protein is released, freeing the RNA polymerase core enzyme.
  5. Once the sigma (σ) factor protein is released, transcription transitions to the elongation phase as the RNA polymerase core enzyme incorporates additional nucleotides to the 3’ end of the RNA transcript.

Key Questions

  • Describe the initiation phase of transcription in bacteria.

Elongation in Bacteria

The elongation phase of transcription in bacteria involves RNA synthesis by the RNA polymerase core enzyme (see figure 9.5). The E. coli RNA polymerase core enzyme has the following features:

EDI_9.5_Transcription_Elongation-01_1.jpg
Figure 9.5 Transcription Elongation --- Image used from OpenStax (access for free at https://books.byui.edu/-vuzA)

Key Questions

  • What are the similarities between the RNA polymerase core enzyme and the DNA polymerases discussed previously?
  • What are the differences between the RNA polymerase core enzyme and the DNA polymerases discussed previously?
  • Which protein functions as the helicase for transcription?
  • Which molecules provide the energy for transcription?

Transcription of Multiple Genes

Not all genes use the same DNA strand as the template strand. In figure 9.6, genes A and B use the bottom DNA strand as the template strand for RNA synthesis, while gene C uses the top DNA strand as the template strand. Genes A and B are transcribed left to right, while gene C is transcribed right to left.  Notice that the promoters for genes A and B are to the left of the gene, while the promoter for gene C is to the right of the gene.

8.6_Transcription_of_Multiple_Genes.jpg
Figure 9.6 Transcription of Multiple Genes --- Image created by SL

Rho (ρ)-Dependent Termination

While the RNA polymerase core enzyme is synthesizing RNA, an RNA-DNA double helix molecule is formed within the active site of the enzyme. Transcriptional termination involves the weakening of the hydrogen bonds within this RNA-DNA double helix, resulting in dissociation of the RNA and the RNA polymerase core enzyme from the DNA.

Transcriptional termination can occur in two different ways in E. coli:

 The rho (ρ)–dependent mechanism of termination requires binding between the rho (ρ) protein, a helicase that breaks the hydrogen bonds within an RNA-DNA double helix, and an RNA sequence near the 3’ end of the RNA transcript called the rho utilization site (rut) (see figure 9.7). The ρ-dependent mechanism of transcription termination also requires the formation of a secondary structure within the RNA transcript called a stem-loop or hairpin loop. The stem-loop is formed when guanine (G) and cytosine (C) bases are produced in the RNA when RNA polymerase reads the terminator DNA sequence. The stem-loop, composed of hydrogen bonds between these G and C nucleotides within the same RNA molecule, slows the RNA polymerase core enzyme down during transcription. The ρ protein then catches up with the RNA polymerase, separates the RNA from the template DNA strand within the active site of the RNA polymerase, and releases the RNA transcript and the RNA polymerase core enzyme from the DNA. Transcription is terminated.

Key Questions

  • What three components are involved in ρ-dependent termination?
  • What are the functions of each of these components in ρ-dependent termination?

Rho (ρ)-Independent Termination

The rho (ρ)-independent mechanism does not require ρ protein or the rut sequence (see figure 9.7). In rho (ρ)-independent termination of transcription, a stem-loop structure is formed in the newly synthesized RNA that slows the RNA polymerase core enzyme. This pausing of the RNA polymerase is aided by another protein called NusA. While the RNA polymerase slows down, a uracil-rich region is generated in the RNA because the RNA polymerase core enzyme is copying an adenine-rich region in the template DNA strand. Each uracil base in the RNA forms two hydrogen bonds with an adenine base in the template DNA strand. This weak base pairing between U and A bases tends to dissociate spontaneously, releasing the RNA and RNA polymerase, terminating transcription.

The mechanism that is used for transcription termination depends on the gene. About 50% of E. coli genes use the ρ-dependent mechanism, the other 50% use the ρ-independent mechanism.

8.7_Transcription_Termination_in_Bacteria.jpg
Figure 9.7 Transcription Termination in Bacteria --- Image created by SL

Key Questions

  • What three components are involved in ρ-independent termination?
  • What are the functions of each of these components in ρ-independent termination?

B. Transcription in Eukaryotes

Transcription is one of the most important biological processes in a eukaryotic cell, as the activation of transcription allows eukaryotic cells to adapt to environmental changes (for example, the presence or absence of a hormone or growth factor). Moreover, many eukaryotic organisms are multicellular, so genes need to be transcribed at the right time during development or in the correct cell type. For example, genes involved in building the central nervous system should be transcribed when the nervous system is forming during development. Genes that encode proteins involved in muscle contraction should be transcribed in muscle cells and not transcribed in other cell types, such as neurons or white blood cells. These phenotypic differences are due to differences in transcription, as all cell types in the body contain an identical collection of genes.

DNA Sequences Control Eukaryotic Transcription

The transcription of eukaryotic genes is controlled by several types of DNA sequence elements, including the following (see figure 9.8):

For a eukaryotic gene to be transcribed, the TATA box and the +1 site must be present. If these two sequences are the only DNA sequences present upstream of a gene, the gene is transcribed at a low, yet constant rate, the so-called basal level of transcription.

The DNA sequences that influence transcription of an adjacent gene are called cis-acting elements. Cis-acting elements include the core promoter, enhancers, and silences. The transcription factor proteins that bind to these cis-acting elements are called trans-acting factors. Trans-acting factors, also called transcription factors, include activator proteins, repressor proteins, and the general transcription factor (GTF) proteins (see below).

EDI_9.8_Eukaryotic_Promoter-01-01_1.jpg
Figure 9.8 Eukaryotic Promoter --- Image created by SL

Key Questions

  • What are the names of the two sequence features within the core promoter?
  • What are the two functions of the TATA box?
  • What are names and functions of the two regulatory DNA sequences that influence the transcription of eukaryotic genes?
  • What are the names of the proteins that bind to these two regulatory DNA sequences?

RNA Polymerases in Eukaryotes

In eukaryotes, there are three types of RNA polymerases that handle transcription:

Key Questions

  • What types of genes do the three eukaryotic RNA polymerases transcribe?

Initiation in Eukaryotes

Both basal (constant, low level) transcription and regulated (transcription above or below the basal level) transcription in eukaryotes require the following proteins (see figure 9.9):

 The association of RNA polymerase II with the six GTF proteins listed above forms a preinitiation complex. The preinitiation complex is also called the basal transcription apparatus.

 

8.9_Transcription_Initiation_in_Eukaryotes.jpg
Figure 9.9 Transcription Initiation in Eukaryotes --- Image used from OpenStax (access for free at https://books.byui.edu/-vuzA)

Key Questions

  • Which of the GTFs binds to the core promoter?
  • Which of the GTFs acts as a bridge to connect the GTF bound to the core promoter to the GTF bound to RNA polymerase II?
  • Which of the GTFs is both a kinase and a helicase?
  • Which GTF activates RNA polymerase II?

General and Regulatory Transcription Factors

Transcription factors are proteins that influence the ability of RNA polymerase II to bind to a eukaryotic core promoter. A huge number of eukaryotic genes encode transcription factor proteins; it is estimated that as many as 1000 human genes encode proteins that regulate transcription!

There are two general categories of transcription factor proteins:

The DNA binding sites (core promoter, enhancers, and silencers) for these transcription factors tend to be near the genes they control. As a result, the DNA sequences are called cis-acting elements.  However, these cis-acting elements do not need to be immediately adjacent to the core promoter. Some enhancers and silencers can be within the gene that they control or can be hundreds of thousands of base pairs away.  The transcription factor proteins (GTFs, activators, and repressors) that bind to the cis-acting elements are called trans-acting factors.

A large multi-subunit protein complex called mediator regulates the interaction between RNA polymerase II and the activator and repressor proteins. Mediator thus serves as a link between transcription factors that bind to enhancers and silencers and RNA polymerase II, thereby determining the overall rate of transcription.

8.10_Regulatory_Transcription_Factors_and_Mediator.jpg
Figure 9.10 Regulatory Transcription Factors and Mediator --- Image created by SL

Key Questions

  • What are three examples of cis-acting elements?
  • What are three examples of trans-acting factors?
  • What is the function of the mediator complex?

Transcription Elongation in Eukaryotes

 The elongation step in eukaryotic transcription is virtually identical to the transcription elongation step in prokaryotes. RNA polymerase II in eukaryotes has the same functional capabilities as the RNA polymerase core enzyme in E. coli.

Key Questions

  • What are the names of the two proteins that act as a DNA helicase in eukaryotic transcription?

Transcription Termination in Eukaryotes

Transcriptional termination in eukaryotes occurs during the process of polyadenylation, a modification to the 3’ ends of mRNAs in eukaryotes. We will cover polyadenylation in more detail in Part 10. In short, a protein called cleavage and polyadenylation specificity factor (CPSF) binds to a sequence called the polyadenylation signal sequence in the mRNA. CPSF then acts as an endonuclease, cleaving the mRNA approximately 20 nucleotides downstream (towards the 3’ end of the mRNA) from the polyadenylation signal sequence. Cleavage of the mRNA by CPSF releases the mRNA from RNA polymerase II.

After CPSF releases the mRNA from RNA polymerase II, there are two potential ways that RNA polymerase II can be released from the DNA, thereby terminating transcription:

8.11_Transcription_Termination_in_Eukaryotes_A_Torpedo_Model.jpg

8.11_Transcription_Termination_in_Eukaryotes_B_Allosteric_Model.jpg
Figure 8.11 Transcription Termination in Eukaryotes A) Torpedo Model B) Allosteric Model --- Images created by SL

Key Questions

  • What is the difference between the torpedo and the allosteric models of transcription termination?

Review Questions

Fill in the blank:

  1. When structural genes are expressed, they produce ___________________molecules; when nonstructural genes are expressed, they produce _________________molecules.
  2. _________________ is an example of a protein that has both helicase and kinase activity.
  3. _____________ binds to the -10 and -35 sequences.
  4. The RNA polymerase holoenzyme consists of the _____________________________ and _________________________.
  5. The -25 sequence is the binding site for the _________________ protein.
  6. The ____________ protein binds to the rut sequence found in 50% of bacterial mRNA molecules.
  7. RNA polymerase _____ is responsible for transcribing eukaryotic structural genes.
  8. Phosphorylation of _________________________ helps to activate transcription in eukaryotes.
  9. A(n) __________________ protein binds to an enhancer sequence in the DNA to activate transcription above the basal level, while a(n) ______________ protein binds to a silencer sequence in the DNA to decrease transcription below the basal level.
  10. The protein ____________ causes the RNA polymerase core enzyme to pause at the stem loop in the rho (ρ)-independent mechanism.
  11. DNA replication requires the use of DNA helicase to unwind double-stranded DNA, while transcription in bacteria uses the _________________________ to unwind double-stranded DNA.

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