GENE EXPRESSION AND REGULATION Molecular Genetics

GENES Genes are small sections of DNA within the genome that code for proteins. They contain the instructions for our individual characteristics – like eye and hair colour.  A typical gene consists of an upstream region (promoter region), coding segments (exons) and non-coding segments (introns)

LEARNING FOCUS Gene expression: the genetic code and roles of RNA in transcription, RNA processing in eukaryotes, and translation Gene expression is the process by which the instructions in our DNA are converted into a functional product, such as a protein. Gene expression is a tightly regulated process that allows a cell to respond to its changing environment. It acts as both an on/off switch to control when proteins are made and also a volume control that increases or decreases the amount of proteins made. There are two key steps involved in making a protein, transcription and translation. 

TRANSCRIPTION and TRANSLATION Link to interactive http://learn.genetics.utah.edu/content/basics/geneanatomy/

GENE EXPRESSION The overall process of gene expression involves two major stages The first stage of gene expression involves transcription, which is 'rewriting' or copying of information from DNA to ribonucleic acid (RNA). The synthesised RNA is complementary to the sequence of one strand of the DNA (the template strand). The other strand is called the coding strand. An important RNA molecule produced during transcription is called messenger RNA (mRNA) because the molecule functions as a messenger, carrying a copy of the code into the cytoplasm.

Synthesising messenger RNA Messenger RNA synthesis is controlled by the enzyme RNA polymerase. A typical gene consists of an upstream region (promoter region), coding segments (exons) and non-coding segments (introns) The promoter region of a gene has a specific sequence recognised by RNA polymerase that initiates transcription.

Synthesising mRNA continued The coded RNA (primary transcript) is modified by enzymes that cut out the regions that corresponded to the introns (non-coding DNA) of the gene and join the remaining pieces back together. This shortened RNA molecule corresponding to the exon (coding) regions of the gene is the messenger RNA (mRNA). The mRNA molecule is complete when its two ends are modified. It is chemically 'capped' at the 5′ end. A tail of As (a poly-A tail) is added at the 3′ end. This is the mRNA that then moves out through the nuclear pores into the cytoplasm.

TRANSLATION – assembling proteins The second stage of gene expression is translation. In this process, the 'instructions' in the mRNA are read and a polypeptide (protein) product is assembled. The mRNA is transported to the ribosomes that are either free in the cytoplasm or located on the rough endoplasmic reticulum. The ribosomes provide a scaffold for the mRNA to assemble. At the ribosomes, the sequence of bases in the mRNA is 'decoded' to give the sequence of amino acids of the polypeptide.

GENE REGULATION Gene regulation involves processes that control gene expression, turning a particular gene 'on' or 'off'. Your body only transcribes genes and produces proteins when they are needed in order to save energy and resources. All the somatic cells in your body contain the same chromosomes and therefore the same DNA and same genes. However, these cells are able to have different shapes and sizes and perform different functions and change throughout your lifespan. These differences are possible because of different mechanisms that control the expression of individual genes. These mechanisms are collectively referred to as mechanisms for gene regulation.

GENE REGULATION There are many steps involved in the expression of gene, therefore there are many different mechanisms for regulating expression: The structure of genes varies The rate of transcription can be regulated Post-transcriptional modifications can influence which protein is produced The rate of translation can be regulated The activity of the protein product (enzyme) can be regulated Genes can also be regulated by their environment Light, Temperature, Ions, Hormones Gene structure All genes contain an upstream promoter region. This consists of a binding site for RNA polymerase and other base sequences known as upstream promoter elements (UPEs). UPEs initiate transcription. Genes vary in the number and type of UPEs. A gene with only one UPE will be weakly expressed. A gene with many UPEs is actively transcribed. Other DNA sequences known as enhancers increase the rate of transcription. Genes which code for the production of essential proteins are often present as multiple copies. Genes can be permanently inactivated in some cells by changes in the chromosome’s structure. Transcription rate DNA binding proteins called transcription factors, regulate the rate at which a gene is transcribed. These proteins bind with the upstream region of the gene and stimulate transcription. Transcription factors may be activated by hormones. Post-transcriptional modifications Some pre-mRNAs can be modified in more than one way. Pre-mRNA may be spliced differently in different tissues, leading to different protein products. Translation Cells can regulate the amount of translation which occurs by controlling the life-span of mRNA; mRNA may be inactivated after only a short time being translated, or may survive longer in the cell and be translated many times. Protein activity Gene expression may be regulated by controlling the activity of the proteins produced in translation. For example, enzyme inhibitors may inactivate an enzyme until it is needed. Some proteins may control the production of other proteins – e.g. repressor proteins can bind to promoter region of DNA and prevent transcription.

GENE REGULATION Gene regulation is not as well understood in multicellular organisms such as plants and mammals as it is in bacteria, but similar processes do apply. To understand this we are going to look at how the lactose gene is turned on and off in E Coli.

lac operon An operon is a group of genes that are all transcribed at the same time. The lac operon consists of three genes each involved in processing the sugar lactose. One of them is the gene for the enzyme β-galactosidase This enzyme hydrolyses lactose into glucose and galactose

Bacteria adapting to its environment E. coli can use either glucose, which is a monosaccharide, or lactose, which is a disaccharide for energy. However, to use lactose it needs to be hydrolysed (digested) first. So the bacterium prefers to use glucose when it can.

Four situations are possible When glucose is present and lactose is absent the E. coli does not produce β-galactosidase. When glucose is present and lactose is present the E. coli does not produce β-galactosidase. When glucose is absent and lactose is absent the E. coli does not produce β-galactosidase. When glucose is absent and lactose is present the E. coli does produce β-galactosidase

When lactose is absent A repressor protein is continuously synthesised. It sits on a sequence of DNA just in front of the lac operon, the Operator site The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing Regulator gene lac operon Operator site z y a DNA I O Repressor protein RNA polymerase Blocked

When lactose is present A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site z y a DNA I O Promotor site z y a DNA I O

Repressor protein removed When both glucose and lactose are present This explains how the lac operon is transcribed only when lactose is present. BUT….. this does not explain why the operon is not transcribed when both glucose and lactose are present. When glucose and lactose are present RNA polymerase can sit on the promoter site but it is unstable and it keeps falling off Promotor site z y a DNA I O Repressor protein removed RNA polymerase

Activator protein steadies the RNA polymerase When glucose is absent and lactose is present Another protein is needed, an activator protein. This stabilises RNA polymerase. The activator protein only works when glucose is absent In this way E. coli only makes enzymes to metabolise other sugars in the absence of glucose Promotor site z y a DNA I O Transcription Activator protein steadies the RNA polymerase

Lifted off operator site Sits on the promoter site Summary Carbohydrates Activator protein Repressor protein RNA polymerase lac Operon + GLUCOSE + LACTOSE Not bound to DNA Lifted off operator site Keeps falling off promoter site No transcription - LACTOSE Bound to operator site Blocked by the repressor - GLUCOSE Bound to DNA Sits on the promoter site Transcription