Ch. 11 - Transcription of Genes

Question 1

Strands of DNA

Answer 1

Antisense/template strand: Strand of DNA used as a guide for synthesizing a new strand by complementary base pairing.

Sense/coding strand: The strand of DNA equivalent in sequence to the mRNA (same as plus strand).

Question 2

Housekeeping/constitutive genes

Answer 2

Genes required for the fundamental operations of the cell that are expressed during most/all conditions. Needed for essential life functions.
Other genes vary in expression in response to changes in the environment.

Question 3

Cistrons and Structural genes

Answer 3

Cistron: Segment of DNA (or RNA) that encodes a single polypeptide chain.
Structural gene: Sequence of DNA (or RNA) that encodes for a protein or for an untranslated RNA molecule (rRNA, tRNA, snRNA (small nuclear RNA), etc.)

Monocistronic mRNA: In eukaryotes, each mRNA carries the information from only a single gene. However, the gene can contain multiple exons and lead to the production of several proteins due to alternative splicing.

Polycistronic mRNA: In prokaryotes, several genes may be carried on the same mRNA in operons (clusters of related genes, regulated by the same regulatory regions, yielding a single polycistronic mRNA molecule). The mRNA molecule carries information of multiple genes, which are coding sequences for several proteins.

Question 4

Open Reading Frame (ORF)

Answer 4

Any sequence of bases that could theoretically encode a protein. Must have a start and stop coding that are “in frame” (sequence of bases can be divided by three to make codon triplets).
A cistron that encodes a protein is an ORF, whereas a cistron that encodes an untranslated RNA is not an ORF.

Question 5

The structure of a gene

Answer 5

Upstream of the gene are the regulatory regions and the promoter. The regulatory regions bind transcription factors that either repress or activate transcription. The promoter is the region, in front of the gene, where the RNA polymerase binds, and promotes the expression of the gene.

After the promoter region, is the transcription start site (TSS). TSS occurs at nucleotide +1. Between the promoter and the start codon in the ORF/gene is the 5’-UTR (containing a ribosome binding site for translation).

In prokaryotes, there can be several genes organized in an operon. In eukaryotes, each gene usually contains coding parts called exons and non-coding parts called introns.

Downstream of the gene is the transcription terminator. Between the stop codon in the ORF/gene is the 3’-UTR.

Question 6

RNA polymerase and the promoter

Answer 6

RNA polymerase is a holoenzyme consisting of two main components, the core enzyme (responsible for the RNA synthesis) and the sigma subunit (responsible for the recognition of the promoter). Different RNA polymerases have different sigma subunits, the most common being the sigma70 subunit. This sigma subunit recognizes two special sequences in the promoter of the sense strand: the -10 region (TATA box) and the -35 region.

In prokaryotes, the core enzyme consists of 5 subunits: alfa I, alfa II, beta, beta marked and w. The beta-subunits comprise the catalytic part of the enzyme. The alfa- and w-subunits are help assemble the core enzyme.
Eukaryotes and Archaea have RNA polymerases with additional subunits.

Question 7

Transcription initiation

Answer 7

Once the sigma subunit of RNA polymerase has bound to the promoter, the RNA polymerase core enzyme opens up the DNA double helix locally and temporarily to form the transcription bubble.

The RNA polymerase synthesizes an mRNA strand, using the antisense strand as a template for complementary base pairing. Synthesis of 6-9 bases, usually starting with an A (U in mRNA) occur before the RNA polymerase moved from the promoter. When the core enzyme of the RNA polymerase then moves along the DNA, the sigma subunit either detaches from the DNA or is left at the promoter.

When RNA polymerase progresses along the DNA, it unwinds the DNA and produce positive supercoils. These are released by DNA gyrase, which inserts negative supercoils ahead of RNA polymerase. Topoisomerase removes negative supercoils behind the RNA polymerase.

Question 8

Transcription termination

Answer 8

The end of a gene is marked/has a region called the terminator sequence that forms a hairpin structure in the mRNA. The terminator is in the template strand of DNA, consisting of two inverted repeats separated by approx. 10 bases (the loop), followed by a run of adenines. This gives a hairpin structure in the mRNA followed by a run of uracils at the 3’ end. RNA polymerase pauses when it reaches the hairpin structure.

The stem loop destabilizes the interaction of the mRNA and the RNA polymerase, and weakens the binding between the mRNA and DNA as AU base pairs are weaker (only two H-bonds). The combination of these two creates instability in the complex, resulting in mRNA and DNA release from the RNA polymerase, and transcription is terminated.

Question 9

Termination and Rho

Answer 9

There are two classes of terminators: Rho-dependent terminators and Rho-independent terminators.

Rho-dependent terminators need the recognition protein Rho to separate the RNA polymerase from the DNA, while Rho-independent terminators do not need Rho or any other factor to cause termination.

Rho-proteins recognizes and bind to their recognition site in the RNA, causing allosteric changes in the catalytic subunit of RNA polymerase that results in termination. Rho unwinds the mRNA/DNA helix in the transcription bubble, and separates the two strands, leading to disassembly.

Question 10

Regulatory proteins

Answer 10

Activators: binds to the promoter region and facilitates binding of the sigma subunit of RNA polymerase. Thus inducing transcription of the gene.

Repressor: binds to the operator, which often overlaps with the promoter. This blocks parts of the promoter, and prevents RNA polymerase from binding to it. Thus, inhibiting transcription of the gene.

Positive regulation of genes: when an activator is bound to induce expression.
Negative regulation of genes: when a repressor is bound to repress expression.

Recognition sites on DNA are often inverted repeats. Separate subunits of the regulatory protein each bind one of the repeat sequences.

Question 11

Co-inducers and -repressors

Answer 11

Some DNA-binding regulatory proteins are allosteric, and are activated or inhibited by binding of signal molecules. This occurs because of changes in their conformation.

Co-inducers: binds to a regulatory protein to activate transcription.
Co-repressor: signal molecule that activates a repressor, inhibits transcription.

Question 12

Gene regulation in eukaryotes

Answer 12

Eukaryotic gene regulation is much more complex than for prokaryotic. In prokaryotes, transcription is tightly followed by translation. The separation of the nucleus and the cytosol makes it possible to separate transcription and translation. This also allows mRNA to be modified before it leaves the nucleus.
Modification gives several opportunities when it comes to regulation.

Eukaryotes have gene regulation on different levels (7 in biochemistry):

• DNA, chromosomes: structure, packing, methylation, acetylation, euchromatin
• RNA transcript: transcription factors
• Functional RNA: modification of the primary transcript, alternative splicing, 5’ cap, poly-A tail
• Pre-translation: degradation, turnover, transport of mRNA
• Translation, ribosome binding, tRNA
• Protein: modification, degradation
• Active protein/Inactive protein: modification, acetylation

Question 13

Eukaryotic RNA polymerases

Answer 13

RNA polymerase I: transcribes the genes for the two large rRNA molecules.
RNA polymerase III: transcribes the genes for tRNA, 5S rRNA, some snRNA, and several other RNA molecules.
RNA polymerase II: transcribes most eukaryotic genes that encode proteins, and as a result is subject to the most complex regulation.
RNA polymerase I and III operate constitutively in most cell types, as most RNA molecules are needed at all times.

Plants have two additional RNA polymerases (IV and V). In addition, mitochondria and chloroplasts have bacterial RNA polymerases.

Question 14

General and specific transcription factors

Answer 14

There is a diversity of many different transcription factors that contribute to the transcriptional complex. About 1500 genes code for regulatory proteins that are transcription factors.

General/Basal: needed for all genes transcribed by a given RNA polymerase

Specific: required for the transcription of particular genes under given conditions.

Question 15

rRNA gene clusters

Answer 15

The genes for rRNA are located at multiple sites along the DNA. The 45S RNA precursors are processed to produce the 18S and 28S subunit of rRNA. A lot of genes code for rRNA. In eukaryotic cells, several hundred rRNA genes are localized in clusters. Upstream Binding Factor 1 (UBF1) recognizes the GC rish region in the core promotor in rRNA genes, and activates transcription of rRNA genes.

Question 16

Transcription of eukaryotic genes

Answer 16

RNA polymerase transcribes most protein coding genes, where promoter recognition and initiation of transcription requires the general transcription factors. Some of the specific transcription factors bind to enhancer sequences/regions, which may be located thousands of bp from the promoter.

Promoters of eukaryotic genes can be divided into three regions.
The initiator box is where the transcription starts (+1).
The TATA box is located -25 bp upstream of the initiator box, and contain an AT-rich region. The TATA region is recognized by the TATA binding protein (TBP), and is needed for the binding of RNA polymerase.
Upstream elements are located 50-200 bp upstream of the start site.

Question 17

Transcription initiation in eukaryotes

Answer 17

TBP is a part of a protein complex known as TFIID and is the first step required for the binding of RNA polymerase II. Additional transcription factors needed are:
TFIIB which binds adjacent to TFIID
TFIIH which pry apart the DNA double helix, exposing the template strand.
TFIIA and TFIIJ binds to the complex
In the last step, TFIIF and the RNA polymerase II bind to the complex.

After all the general transcription factors are bound, TFIIH needs to phosphorylate the Ser and Thr residues at the tail (carboxyl-terminal domain, CTD) of the RNA polymerase II, before RNA synthesis is initiated.

After the phosphorylation, the other general transcription factors, except TFIIH, dissociate, and RNA synthesis is initiated.

Question 18

Upstream elements

Answer 18

Upstream elements and the binding of their associated specific transcription factors are needed for efficient transcription of genes. Upstream elements are recognition sites for many specific transcription factors, which usually make contact with the transcription apparatus through TFIIA, TFIIB, and TFIID. The general transcription factors facilitate assembly of the transcription complex.

Question 19

Regulation of RNA polymerase II

Answer 19

RNA polymerase II can be subject to negative regulation by negative elongation factor (NELF) and DRB-sensitive inducing factor (DSIF), which bind to RNA polymerase II and cause it to pause transcription. Transcription resumes when NELF, DSIF, and the CTD domain of RNA polymerase are phosphorylated.

Question 20

Enhancers

Answer 20

Enhance transcription by binding specific transcription factors. An enhancer can contain a cluster of recognition sites, and are often located thousands of base pairs away from the gene they control.

Enhancers can be located both upstream and downstream of the gene. Transcription factors that bind to enhancers interacts with the transcription complex, causing the DNA to bend in large loops.