The genome contains the hereditary information of the structure and function of a cell or organism. This information is stored as a sequence of bases in DNA. A relatively small percentage of DNA codes for proteins and ribonucleic acids (RNAs), while a large amount of the genome is composed of sequences without a clear function. The conversion of the information stored within DNA into a functional molecule, or RNA and proteins, is termed gene expression. Gene expression occurs in two stages: transcription and translation. During transcription, DNA is copied into RNA. RNA is then used to synthesize proteins during translation.
Key enzymes involved in transcription are DNA-dependent RNA polymerases. These enzymes synthesize the RNA molecule based on the genes encoded in DNA, which contain starting sites (promoters) where transcription begins. Transcription factors are required to recognize the promoter. RNA polymerase moves along the template strand of the double-stranded DNA. The strand is synthesized until the end of the DNA segment (termination site) is reached. In eukaryotes, the newly formed primary transcript is further modified to be, for example, available for protein synthesis.
Gene expression is strongly regulated at all levels. Some genes are expressed in all cells and are required as housekeeping genes for basic cellular functions (i.e., constitutive expression). Other genes are only active in certain cells; their expression is regulated by a variety of mechanisms. Genes can undergo activation or silencing, and transcription depends on the presence of specific DNA-binding proteins. The newly formed RNA may also be degraded after transcription by various mechanisms before use in protein synthesis. There are also regulatory mechanisms at a translational level. Although each cell in an organism contains the same DNA, the regulated expression of certain genes causes the cells to specialize and assume different functions, e.g., muscle cells or hepatocytes.
- Gene expression: conversion of genetic information stored in DNA into a functional gene product (RNA and proteins)
- Protein synthesis: process of gene expression (comprised of transcription and translation) as well as post-transcriptional modifications (see the learning card on translation and protein synthesis for more information)
- Central dogma of molecular biology: genetic information always flows in one direction from DNA to RNA to the protein
- Sense strand: the DNA strand from which the complementary RNA strand is produced
- Antisense strand: the DNA strand complementary to the sense strand
- Specific DNA sequence located upstream (= in the 5' region) of a gene that regulates transcription
- Contains AT-rich sequences, e.g. TATA box and CAAT box
- Binding site for RNA polymerase II and transcription factors at the start of transcription
- Mutations at the site of promoters usually lead to dramatically decreased transcription rate
- Exon-intron structure: eukaryotic genes are composed of alternating coding and noncoding regions
- Substrates: the nucleoside triphosphates ATP, GTP, CTP, and UTP
- Enzymes: RNA polymerases
- General transcription factors: specific helper proteins that help RNA polymerase find and bind to the promoter and initiate RNA synthesis
Transcription reactions are catalyzed by (DNA-dependent) RNA polymerases. In eukaryotic cells, there are various types of RNA polymerase. They recognize different promoter types and transcribe different types of genes.
- Structure: composed of two large subunits with many polypeptide chains
- Function: synthesis of a new RNA strand from 5' to 3' direction; reading of the DNA strand from 3' to 5' direction
|Type of RNA polymerase||Transcripts||Location|
- General transcription factors: enable binding of RNA polymerase to the proximal promoter regions by binding of chromosomal DNA to specific base sequences → start of transcription
- Specific transcription factors: modulate transcription by binding to regulatory elements (enhancers, silencers)
Proteins, such as transcription factors that bind to DNA, require specific protein domains, also termed structural motifs. These structural motifs usually use either an α-helix or a β sheet to bind to the major groove of DNA. Transcription factors have DNA-binding domains through which they are able to interact with specific DNA segments to perform their function. Numerous structural motifs of DNA-binding domains have been identified. Important examples are the zinc finger domains, leucine zippers, basic helix-loop-helix, and the homeobox.
- Zinc finger
- DNA binding: The DNA-binding hydrophilic regions of α-helices contain many basic residues that interact with the major groove of DNA.
- Basic helix-loop-helix
- Homeobox (with helix-turn-helix)
Transcription is divided into three phases: initiation, elongation, termination.
Initiation (transcription): the start of transcription by the formation of the initiation complex and unwinding of DNA
- Preinitiation complex (RNA polymerase-promoter closed complex) formation by binding of general transcription factors and RNA polymerase to the promoter region (e.g., TATA box, CAAT box, GC box)
- Formation of a transcription bubble by unwinding the DNA double helix to a single strand with a length of 10–12 bases (open complex)
- Start of RNA synthesis
- Elongation: extension of the RNA strand
- Termination: During termination, starts.
In eukaryotes, the end-product of transcription is heterogeneous nuclear RNA (hnRNA), which is then transformed into mature mRNA through post-transcriptional modifications in the nucleus. These modifications include capping, polyadenylation, splicing, and RNA editing. mRNA then leaves the nucleus and enters the cytosol
- Definition: addition of a cap of 7-methylguanosine to the 5' end of hnRNA
- Cleavage of the 5'-phosphate group by RNA triphosphatase
- Addition of a GMP residue (formed from GTP with cleavage of pyrophosphate) to the 5' diphosphate end of hnRNA by guanylyltransferase
- Methylation of one, two, or three ribosome residues of hnRNA with S-adenosylmethionine (SAM) as a methyl group donor
- Protects against degradation (through exonucleases )
- Initiation of translation
- Definition: addition of a tail of ∼200 adenosine monophosphates (polyadenylate, A) to the 3' end of hnRNA
- ↑ Stability (protects against degradation)
- Initiates translation
- Definition: excising of introns from hnRNA transcripts and direct linkage of exons
- Function: excision of introns so that the resulting mRNA only contains relevant information in the form of exons
Spliceosome formation at the exon-intron border
- Complex of:
- Involved sequence segments on the hnRNA:
- Opening of the exon-intron border at the 5' splice site: A temporary lariat structure with a 2'→ 5' phosphodiester bond is formed, which links the two ends to be joined together in close proximity (loop formation)
- Opening of the exon-intron border at the 3' splice site
- Joining of the exon ends
- Spliceosome formation at the exon-intron border
- Definition: alteration of RNA base sequences by the insertion, deletion, or modification of individual bases (independent of splicing)
- Function: possibility of producing various proteins
- A-to-I editing: adenosine is deaminated to inosine, i.e., the base adenine is converted to hypoxanthine
C-to-U editing: Cytidine is deaminated to uridine, i.e., the base cytosine is converted to uracil
- Occurs in mRNA
- Typical example of C-to-U editing
- The mRNA for apolipoprotein B (apoB) codes for apoB-100.
- After editing, the mRNA for apoB codes for a markedly smaller protein, apoB-48, because the deamination of cytidine to uridine generates a stop codon through cytidine deaminase.
- Via C-to-U-editing, e.g., apoB-48 is formed by enterocytes compared to apoB-100 by hepatocytes.
- Definition: removal of introns within hnRNA with differential joining of exons
- Process: similar to splicing with additional splicing factors that determine the range of splice locations
- Various proteins can be produced from one gene: increased information density of DNA
- The formation of new proteins is facilitated: more rapid adaptation to altered living conditions.
Quality control of mRNA
Prokaryotic gene regulation (operon model)
Regulation of gene expression was initially analyzed in E. coli. Regulatory sequences in the bacterial genome ensure gene expression of the enzyme β-galactosidase if the sugar lactose is available as an energy source. Other proteins are also synthesized, which are associated with lactose metabolism. Therefore, it involves the coordinated expression of several genes.
- Definition: a model for describing the gene regulatory mechanism in prokaryotes
- Function: adapt to changing environmental conditions by simultaneously increasing the expression of certain related genes
Example: lac operon
- Description: A transcriptional unit of genes for enzymes involved in lactose metabolism that is only expressed in the presence of lactose (e.g. β-galactosidase).
Components (in their order in the genome)
- Regulatory gene lacI: does not directly belong to the lac operon but codes for a repressor protein that binds to the lac operator in the absence of lactose and prevents transcription
- Promoter: binding site for catabolite activator protein (CAP) and RNA polymerase in transcription
- Operator: Binding site of the repressor that overlaps with the promoter.
- lacZ: β-galactosidase gene
- lacY: permease gene
- lacA: transacetylase gene
- Presence of glucose and absence of lactose → transcription cannot take place
- Absence of glucose and presence of lactose → ↑ transcription
Eukaryotic gene regulation
Regulation of gene expression is significantly complicated in eukaryotes compared to prokaryotes. One reason is due to the difference in size between the genomes of eukaryotes and prokaryotes, with eukaryotes having a significantly larger genome. Another reason is that the DNA in the eukaryotic genome in the nucleus is strongly condensed and packaged as chromatin. As a result, it is less accessible than prokaryotic DNA. However, a common feature of eukaryotes and prokaryotes is the importance of activators and repressors, which bind specific DNA sequences and increase or inhibit gene expression.
Distal regulatory elements: DNA sequences that can affect the transcription rate of a gene (can be located before, behind, or in the middle of the gene they regulate)
- Short DNA sequences ∼ 20 bp in length
- Mainly a palindrome or a tandem repeat
- Increase the transcription rate by binding of specific transcription factors (activators)
Examples of an enhancer: hypoxia-response element (HRE)
- The transcription factor hypoxia-inducible factor (HIF) binds to the HRE sequence during hypoxia and induces certain target genes that are important in the response to hypoxia, e.g., expression of EPO and VEGF.
- In normoxia (sufficient amount of oxygen), HIF is hydroxylated by HIF prolyl hydroxylase. Hydroxy-HIF is ubiquitinylated and degraded in the proteasome and is unable to increase the expression of its own target genes.
- Silencer: decreases the transcription rate by binding of specific transcription factors (repressors)
|Actinomycin D|| |