Global Regulatory Mechanisms

Transcription in Prokaryotes

The genetic code is a degenerate, non-overlapping set of 64 codons that encodes for 21 amino acids and 3 stop codons.

Learning Objectives

Describe the genetic code and how the nucleotide sequence prescribes the amino acid and the protein sequence

Key Takeaways

Key Points

  • The relationship between DNA base sequences and the amino acid sequence in proteins is called the genetic code.
  • There are 61 codons that encode amino acids and 3 codons that code for chain termination for a total of 64 codons.
  • Unlike, eukayrotes, a bacterial chromosome is a covalently-closed circle.
  • The DNA double helix must partially unwind for transcription to occur; this unwound region is called a transcription bubble.

Key Terms

  • nucleotide: the monomer comprising DNA or RNA molecules; consists of a nitrogenous heterocyclic base that can be a purine or pyrimidine, a five-carbon pentose sugar, and a phosphate group
  • amino acid: Any of 20 naturally occurring α-amino acids (having the amino, and carboxylic acid groups on the same carbon atom), and a variety of side chains, that combine, via peptide bonds, to form proteins.
  • redundancy: duplication of components, such as amino acid codons, to provide survival of the total system in case of failure of single components

The Genetic Code: Nucleotide sequences prescribe the amino acids

The genetic code is the relationship between DNA base sequences and the amino acid sequence in proteins. Features of the genetic code include:

  • Amino acids are encoded by three nucleotides.
  • It is non-overlapping.
  • It is degenerate.

There are 21 genetically-encoded amino acids universally found in the species from all three domains of life. ( There is a 22nd genetically-encooded amino acid, Pyl, but so far it has only been found in a handful of Archaea and Bacteria species.) Yet there are only four different nucleotides in DNA or RNA, so a minimum of three nucleotides are needed to code each of the 21 (or 22) amino acids. The set of three nucleotides that codes for a single amino acid is known as a codon. There are 64 codons in total, 61 that encode amino acids and 3 that code for chain termination. Two of the codons for chain termination can, under certain circumstances, instead code for amino acids.

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Genetic Code Table.: A codon is made of three nucleotides. Consequently there are 43 (=64) different codons. The 64 codons encode 22 different amino acids and three termination codons, also called stop codons.

Degeneracy is the redundancy of the genetic code. The genetic code has redundancy, but no ambiguity. For example, although codons GAA and GAG both specify glutamic acid (redundancy), neither of them specifies any other amino acid (no ambiguity). The codons encoding one amino acid may differ in any of their three positions. For example, the amino acid glutamic acid is specified by GAA and GAG codons (difference in the third position); the amino acid leucine is specified by UUA, UUG, CUU, CUC, CUA, CUG codons (difference in the first or third position); while the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC (difference in the first, second or third position). These properties of the genetic code make it more fault-tolerant for point mutations.

Origin of transcription on prokaryotic organisms

Prokaryotes are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. The central region of the cell in which prokaryotic DNA resides is called the nucleoid region. Bacterial and Archaeal chromosomes are covalently-closed circles that are not as extensively compacted as eukaryotic chromosomes, but are compacted nonetheless as the diameter of a typical prokaryotic chromosome is larger than the diameter of a typical prokaryotic cell. Additionally, prokaryotes often have abundant plasmids, which are shorter, circular DNA molecules that may only contain one or a few genes and often carry traits such as antibiotic resistance.

Transcription in prokaryotes (as in eukaryotes) requires the DNA double helix to partially unwind in the region of RNA synthesis. The region of unwinding is called a transcription bubble. Transcription always proceeds from the same DNA strand for each gene, which is called the template strand. The RNA product is complementary to the template strand and is almost identical to the other (non-template) DNA strand, called the sense or coding strand. The only difference is that in RNA all of the T nucleotides are replaced with U nucleotides.

The nucleotide on the DNA template strand that corresponds to the site from which the first 5′ RNA nucleotide is transcribed is called the +1 nucleotide, or the initiation site. Nucleotides preceding, or 5′ to, the template strand initiation site are given negative numbers and are designated upstream. Conversely, nucleotides following, or 3′ to, the template strand initiation site are denoted with “+” numbering and are called downstream nucleotides.

The trp Operon: A Repressor Operon

The trp operon is a repressor operon that is either activated or repressed based on the levels of tryptophan in the environment.

Learning Objectives

Explain the relationship between structure and function of an operon and the ways in which repressors regulate gene expression

Key Takeaways

Key Points

  • The operator sequence is encoded between the promoter region and the first trp-coding gene.
  • The trp operon is repressed when tryptophan levels are high by binding the repressor protein to the operator sequence via a corepressor which blocks RNA polymerase from transcribing the trp-related genes.
  • The trp operon is activated when tryptophan levels are low by dissociation of the repressor protein to the operator sequence which allows RNA polymerase to transcribe the trp genes in the operon.

Key Terms

  • repressor: any protein that binds to DNA and thus regulates the expression of genes by decreasing the rate of transcription
  • operon: a unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together

The trp Operon: A Repressor Operon

Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon. If tryptophan is present in the environment, then E. coli does not need to synthesize it; the switch controlling the activation of the genes in the trp operon is turned off. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized.

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The trp operon: The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed.

A DNA sequence that codes for proteins is referred to as the coding region. The five coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site. Each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription.

A DNA sequence called the operator sequence is encoded between the promoter region and the first trp-coding gene. This operator contains the DNA code to which the repressor protein can bind. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding and transcribing the downstream genes.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.

The Stringent Response

The stringent response is a stress response that occurs in bacteria in reaction to amino-acid starvation or other stress conditions.

Learning Objectives

Explain the function of the alarmone (p)ppGpp in the stringent response

Key Takeaways

Key Points

  • The stringent response is signaled by the alarmone (p)ppGpp.
  • In Escherichia coli, (p)ppGpp production is mediated by the ribosomal protein L11 and the ribosome-associated protein RelA.
  • In other bacteria, stringent response is mediated by a variety of RelA/SpoT Homologue (RSH) proteins. Some only have synthetic, hydrolytic, or both (Rel) activities.

Key Terms

  • stringent response: The stringent response, also called stringent control, is a stress response that occurs in bacteria and plant chloroplasts in reaction to amino-acid starvation, fatty acid limitation, iron limitation, heat shock, and other stress conditions.
  • alarmone: Alarmone is an intracellular signal molecule that is produced due to harsh environmental factors.
  • amino-acid starvation: The amino acid response pathway is triggered by a shortage of any essential amino acid.

The stringent response, also called stringent control, is a stress response that occurs in bacteria and plant chloroplasts in reaction to amino-acid starvation, fatty acid limitation, iron limitation, heat shock, and other stress conditions. The stringent response is signaled by the alarmone (p)ppGpp and modulating transcription of up to 1/3 of all genes in the cell. This in turn causes the cell to divert resources away from growth and division and toward amino acid synthesis in order to promote survival until nutrient conditions improve.

In Escherichia coli, (p)ppGpp production is mediated by the ribosomal protein L11. The ribosome-associated protein RelA with the A-site bound deacylated tRNA is the ultimate inducer. RelA converts GTP and ATP into pppGpp by adding the pyrophosphate from ATP onto the 3′ carbon of the ribose in GTP releasing AMP. pppGpp is converted to ppGpp by the gpp gene product, releasing Pi. ppGpp is converted to GDP by the spoT gene product, releasing pyrophosphate (PPi). GDP is converted to GTP by the ndk gene product. Nucleoside triphosphate (NTP) provides the Pi. It is converted to nucleoside diphosphate (NDP).

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An amino acid: The generic structure of an alpha amino acid in its un-ionized form.

In other bacteria, stringent response is mediated by a variety of RelA/SpoT Homologue (RSH) proteins, with some having only synthetic, hydrolytic, or both (Rel) activities. The disable of stringent response by distruption of relA and spoT in Pseudomonas aeruginosa, produces infectious cells and biofilms that have nutrient limitations. They are more susceptible to antibiotics.

During the stringent response, (p)ppGpp accumulation affects the resource-consuming cell processes replication, transcription, and translation. (p)ppGpp is thought to bind RNA polymerase and alter the transcriptional profile, decreasing the synthesis of translational machinery (such as rRNA and tRNA), and increasing the transcription of biosynthetic genes. Additionally, the initiation of new rounds of replication is inhibited and the cell cycle arrests until nutrient conditions improve. Translational GTP involved in protein biosynthesis are also affected by ppGpp, with Initiation Factor 2 (IF2) being the main target.

Chemical reaction catalyzed by RelA: [latex]\text{ATP} + \text{GTP} \rightarrow \text{AMP} + \text{pppGpp}[/latex]

Chemical reaction catalyzed by SpoT: [latex]\text{ppGpp} \rightarrow \text{GDP} + \text{PPi}[/latex]

Repression of Anabolic Pathways

Repression of anabolic pathways is regulated by altering transcription rates.

Learning Objectives

Differentiate between inducible and repressible systems in gene regulation

Key Takeaways

Key Points

  • Regulation of transcription controls when transcription occurs and how much RNA is created.
  • Gene regulation is either controlled by an inducible system or a repressible system.
  • In prokaryotes, regulation of transcription is needed for the cell to quickly adapt to the ever-changing outer environment.

Key Terms

  • anabolic pathways: Anabolism describes the set of metabolic pathways that construct molecules from smaller units.
  • transcription: The synthesis of RNA under the direction of DNA.
  • gene: A unit of heredity; a segment of DNA or RNA that is transmitted from one generation to the next. It carries genetic information such as the sequence of amino acids for a protein.

Repression of anabolic pathways is regulated by altering transcription rates. Transcriptional regulation is the change in gene expression levels by altering transcription rates.

Regulation of transcription controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymerase can be regulated by at least five mechanisms:

  • Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e. sigma factors used in prokaryotic transcription).
  • Repressors bind to non-coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase’s progress along the strand, thus impeding the expression of the gene.
  • General transcription factors position RNA polymerase at the start of a protein -coding sequence and then release the polymerase to transcribe the mRNA.
  • Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA.
  • Enhancers are sites on the DNA helix that are bound to by activators in order to loop the DNA bringing a specific promoter to the initiation complex.

Regulatory protein is a term used in genetics to describe a protein involved in regulating gene expression. Such proteins are usually bound to a DNA binding site which is sometimes located near the promoter although this is not always the case. Sites of DNA sequences where regulatory proteins bind are called enhancer sequences. Regulatory proteins are often needed to be bound to a regulatory binding site to switch a gene on (activator) or to shut off a gene (repressor). Generally, as the organism grows more sophisticated, its cellular protein regulation becomes more complicated and, indeed, some human genes can be controlled by many activators and repressors working together.

In prokaryotes, regulation of transcription is needed for the cell to quickly adapt to the ever-changing outer environment. The presence or the quantity and type of nutrients determines which genes are expressed; in order to do that, genes must be regulated in some fashion. In prokaryotes, repressors bind to regions called operators that are generally located downstream from and near the promoter (normally part of the transcript). Activators bind to the upstream portion of the promoter, such as the CAP region (completely upstream from the transcript). A combination of activators, repressors and rarely enhancers (in prokaryotes) determines whether a gene is transcribed.

Gene regulation can be summarized as how genes respond: inducible systems or repressible systems. An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to “induce expression. ” The manner in which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells. A repressible system is on except in the presence of some molecule (called a corepressor) that suppresses gene expression. The molecule is said to “repress expression. ” The manner in which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.

For example, when E. coli bacteria are subjected to heat stress, the σ32 subunit of its RNA polymerase changes in such a way that the enzyme binds to a specialized set of promoters that precede genes for heat-shock response proteins.

Another example is when a cell contains a surplus amount of the amino acid tryptophan, the acid binds to a specialized repressor protein (tryptophan repressor). The binding changes the structural conformity of the repressor such that it binds to the operator region for the operon that synthesizes tryptophan, preventing their expression and thus suspending production. This is a form of negative feedback.

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Trp Repressor Protein: Here is a diagram of the Trp repressor protein.

In bacteria, the lac repressor protein blocks the synthesis of enzymes that digest lactose when there is no lactose to feed on. When lactose is present, it binds to the repressor, causing it to detach from the DNA strand.

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Annotated Crystal Structure of Dimeric LacI: Annotated crystal structure of dimeric LacI. Two monomers (of four total) cooperate to bind each DNA operator sequence. Monomers (red and blue) contain DNA binding and core domains (labeled) which are connected by a linker (labeled). The C-terminal tetramerization helix is not shown. The repressor is shown in complex with operator DNA (gold) and ONPF (green), an anti-inducer ligand (i.e. a stabilizer of DNA binding).

The AraC Regulator

The L-arabinose operon, also called ara operon, encodes enzymes needed for the catabolism of arabinose to xylulose 5-phosphate.

Learning Objectives

Describe the regulatory mechanism of the AraC protein in the presence and absence of arabinose

Key Takeaways

Key Points

  • The structural gene, which encodes arabinose breakdown enzymes, is araBAD.
  • The ara operon is regulated by the AraC protein.
  • When arabinose is present, arabinose binds AraC and prevents it from interacting.

Key Terms

  • operon: A unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together.
  • L-arabinose: Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group.
  • catabolism: Destructive metabolism, usually includes the release of energy and breakdown of materials.

The L-arabinose operon, also called ara operon, is a gene sequence encoding enzymes needed for the catabolism of arabinose to xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. It has both positive and negative regulation. The operon is found in Escherichia coli (E. coli).

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Arabinose: Structure of arabinose.

It has been a focus for research in molecular biology since 1970, and has been investigated extensively at its genetic, biochemical, physiological, and biophysical levels.

The structural gene, which encodes arabinose breakdown enzymes, is araBAD. The regulator gene is araC. The genes, araBAD and araC, are transcribed in opposite directions.

The operators are araI and araO2. The operators lie between the AraC.

AraI lies between the structural genes and the operator. The araI1 and araI2 are DNA -binding sites that, when occupied by AraC, induce expression.

Sequence of the Operon: 5′—–araC—–araO—–araI—–araB—–araA—–araD—–3′

The ara operon is regulated by the AraC protein. If arabinose is absent, the dimer AraC protein represses the structural gene by binding to araI1 and araO2 and the DNA forms a loop, which prevents RNA polymerase from binding to the promoter of the ara operon, thereby blocking transcription.

When arabinose is present, arabinose binds AraC and prevents it from interacting. This breaks the DNA loop. The two AraC-arabinose complexes bind to the araI site which promotes transcription. When arabinose is present, AraC acts as an activator and it builds a complex: AraC + arabinose. This complex is needed for RNA polymerase to bind to the promoter and transcribe the ara operon.

Also for activation, the binding of another structure to araI is needed: CRP (formerly known as CAP) + cyclic AMP. Thus the activation depends on the presence of arabinose and cAMP.