Regulation of gene expression Regulatory DNA sequences and molecules Gene expression results in a functional gene product ( either RNA or protein) Genes can be constitutive (always expressed, housekeeping genes) or regulated (expressed only under certain conditions in all cells or in a subset of cells) The ability to appropriately express (positive regulation) or repress (negative regulation) genes is essential in all genes Regulation of gene expression occurs primarily at the level of transcription in both prokaryotes and eukaryotes and is mediated through the binding of trans-acting proteins to cis- acting regulatory elements on DNA

Regulatory DNA sequences and molecules A protein transcription factor (trans acting molecule) which regulates a gene on chromosome 6 might itself have been transcribed from a gene on chromosome 11 Some trans-acting factors can negatively affect gene expression The binding of proteins to DNA is through structural motifs such as leucine zipper, or helix-turn-helix in the protein, or zinc finger

Regulation of prokaryotic gene expression In eukaryotes regulation also occurs through modifications to DNA, as well as through posttranscriptional and postranslational events In prokaryotes such as E.coli, the coordinate regulation of genes- whose protein products are required for a particular metabolic pathway- is achieved through operons Operons: groups of genes sequentially arranged on the chromosome along with cis-acting regulatory elements that determine their transcription  single polycistronic mRNA Prokaryotic operon genes are turned on or off as a unit, exhibiting both positive and negative regulation Operon operator is DNA segment that regulates the activity of operon structural genes

Regulation of prokaryotic gene expression If a repressor is bound to the operator  RNA polymerase is blocked and does not produce mRNA  no protein’s made If inducer is present  it binds the repressor  repressor changes shape and does not bind operator If operator is not bound by a repressor  RNA polymerase transcribes protein-coding genes to mRNA  protein’s made The lac operon contains the lacZ, lacY and lacA structural genes, the protein products of which are needed for the catabolism of lactose, when glucose is not available as the preferred fuel lacY gene codes for a permease that facilitates lactose movement into cell lacZ gene codes for  -galactosidase. It hydrolyses lactose to galactose and glucose lacA gene codes for thiogalactoside transacetylase (unkown job)

Lac operon The regulatory portion of the operon is upstream of the 3 structural genes. It consists of the promoter (P) region where RNA polymerase binds, the operator (O) site and the CAP site (catabolite activator protein) where regulatory proteins bind If O site is empty and CAP site is bound by cAMP-CAP complex (CAP: catabolite activator protein or cAMP regulatory protein-CRP-)  lacZ, lacY and lacA genes are expressed Lacl gene- a regulatory gene with its own promoter-codes for the repressor( a trans-acting factor) that binds the operator (O) site Lacl gene is constitutive, its gene product, repressor, is active. Unlike inducible lacZ, lacY and lacA genes, whose expression is co-ordinately regulated

Lac operon When glucose is the only sugar available Operon is repressed (turned off) by the binding of the repressor protein ( the product of the lacl gene) via a helix-turn-helix motif to the operator (O) site, thus preventing transcription (negative regulation)

Lac operon When only lactose is available Lac operon is turned on- as a small amount of lactose is converted to allolactose (inducer)  binds repressor and changes its conformation  repressor can not bind operator Adenylyl cyclase is actively – in the glucose absence- synthesising cAMP  cAMP-CAP complex binds CAP site  RNA polymerase initiates transcription at promoter site  polycistronic mRNA(3 sets of start and stop codons)  its translation produces 3 proteins for lactose use in energy metabolism

Lac operon When both glucose and lactose are available Adenylyl cyclase is deactivated by glucose (catabolite repression)  no cAMP-CAP complex forms  CAP binding site remains empty  RNA polymerase can’t transcribe 3 structural genes (even though repressor is not bound to operator (O) site)

Regulation of eukaryotic gene expression Gene regulation is more complex in eukaryotes. Operons are not present, but coordinate regulation of the transcription of genes located on different chromosomes can be achieved through the binding of trans-acting proteins to cis-acting elements Posttranscriptional regulation at mRNA level include –Alternative mRNA splicing –Control of mRNA stability –Control of translational efficiency Regulation at the protein level occurs by mechanisms that modulate stability, activation or targeting of the protein

mRNA editing Apoprotein B(apoB) is essential component of chylomicrons and VLDL lipoproteins In the intestine only, C residue in CAA codon for glutamine is deaminated to U  UAA stop codon  shorter protein apoB-48 (48% of mRNA) in the intestines, than hepatic apoB-100(full length)

Regulation through modifications to DNA Gene expression in eukaryotes is influenced by DNA availability to transcriptional apparatus, amount of DNA and arrangement of DNA (localised transitions between B and Z forms) 1. Access to DNA: eukaryotic DNA is complexed with histone proteins Euchromatin is transcriptionally active and less condensed and differs from heterochromatin which is more condensed and inactive –Active chromatin contains histone proteins modified at amino terminal ends by acetylation or phosphorylation, decreasing their positive charge and, hence, decreasing their association with negatively charged DNA  This chromatin remodelling relaxes nucleosomes, allowing transcription factors access to DNA

Regulation through modifications to DNA –Methylation in cytosine bases of GC-rich regions upstream of many genes: Transcriptionally active genes are less methylated than their inactive counterparts. Methylation is by methyltransferase using S-adenosylmethionine as methyl donor 2.Amount of DNA: A change in the number of a gene copies gene can affect the amount of gene product produced. Gene amplification- increase in copy number- in response to chemotherapeutic drug such as methotrexate; an inhibitor of dihydrofolate reductase (DHFR)- is required for the synthesis of tetrahydrofolate,.

Gene amplification results in an increase in the number of DHFR genes and resistance to the drug, allowing tetrahydrofolate to be made for DNA synthesis. 3.Arrangement of DNA: allows the generation of different immunoglobulins from a single gene, providing the diversity needed for recognition of an enormous number of antigens Immunoglobulins – eg IgG- consist of 2 light and 2 heavy chains, with each having regions of constant and variable amino acid sequences Variable region is the result of somatic recombination of segments within both light- and heavy-chain genes Regulation through modifications to DNA

4.Mobile DNA elements: Transposons (Tn): a small mobile genetic (DNA) element that moves around the genome or to other genomes within the same cell, usually by copying itself to a second site but sometimes by splicing itself out of its original site and inserting in a new location mediated by transposase, encoded by Tn itself In Direct transposition, transposase cuts out and inserts Tn at a new site. In replicative transposition; Tn copies are inserted elsewhere while original remains in place Transposition is basis for hemophilia A, muscular destrophy, and antibiotic resistance ( receiving plasmids carrying transposons with antibiotic resistance genes