Controlling the genes Lecture 15 pp 267-280

Common Sense There are estimated to be 25,000 genes within the human genome Between 30-60% are active in any one cell type at any one time These include genes for living These include genes for tissue specificity What about the rest - turned off! How and what controls this?

To be or not to be active DNA regulated by DNA? DNA regulated by RNA? DNA regulated by proteins? DNA IS REGULATED BY PROTEINS HOW? Read on…….

Gene Expression Nearly all human cells have a nucleus (not red blood cells) Almost all these nucleated cells have all 23 pairs of chromosomes (actually 22 almost exact pairs and one or more sex chromosomes) However, not all the genes in all these cells are active all the time GENE EXPRESSION - the regulation of which genes are expressed when - is the process by which a gene's DNA sequence is converted into the structures and functions of a cell.

Across the board Bacterial cells exhibit control of gene expression - not all the enzymes needed for metabolism are expressed at all times - just those for the nutrients present in the environment at that time Multicellular organisms exhibit even more elaborate gene expression - we have brain cells, liver cells, kidney cells, etc. that produce different sets of proteins from different genes. We also have the same cell type change which genes it expresses with time - e.g. white blood cells when they start to produce antibody

Common genes If one compares the genes that different cell types express, one finds the following: Housekeeping genes (histones, polymerases, DNA repair, glycolysis, etc) are commonly expressed by all cell types Specialized genes - these are produced only by certain cell types and not others (antibody genes)

Typical expression In humans we estimate that we have about 25,000 genes on the 23 chromosome pairs A differentiated human cell only expresses between 8,000 and 16,000 of its genes Different genes can also be expressed at different levels at different times These differences lead to different types, sizes, function, and morphologies of cells

Expression switching Specialized cells can alter their expression patterns if subjected to external signals Liver cells respond quickly to levels of different enzymes in the blood. Good example in your textbook at the top of page 270 - (may be on the exam!)

Gene Expression Control We learnt recently that Proteins are made from mRNA, which is itself made from DNA - central DOGMA of biology Any one of the steps along this pathway can be controlled to dictate the presence or absence of the protein We could perform alternative splicing as we saw in the last lecture. We could control how much of the mRNA was transported to the cytoplasm. We could control how much protein was made by the ribosomes. We could even regulate which proteins were activated once they have been made. TRANSCRIPTIONAL CONTROL is the most important and the most seen mechanism - sensible

Gene Expression can be controlled at several different levels 08_03_control.steps.jpg 08_03_control.steps.jpg LEARN THIS FIGURE inside out

Gene Regulatory Proteins Most genes have regions (normally upstream - 5’ direction) which bind regulatory proteins Proteins bind to the regulatory DNA sequences (the promotor and, if present, the enhancer) to activate the transcriptional machine These proteins recognize their target DNA based on many factors, including DNA structure, base sequences, and ionic interactions. These proteins fit extremely well into the major groove of the DNA helix - so much so that these are the tightest and most specific molecular interaction known in biology!

08_04_gene.reg.prot.jpg 08_04_gene.reg.prot.jpg

Homo, finger and zipper Regulatory proteins which interact with DNA can be placed into three important structural motifs Homodomain motif - 3 linked alpha helices of the protein make intimate contact with the DNA Zinc-finger motif - a molecule of zinc stabilizes a alpha helix and a beta sheet structure of the protein. Leucine zipper motif - two alpha helices, each from different protein molecules come together to make contact with the major grooves of the DNA Learn the spelling too!

Repressors & Activators Genes can be regulated by both on switches and off switches Gene repressors turn off or reduce gene expression Gene activators turn on or enhance gene expression Read page 273 for a good account Learn what an operon is here - a set of genes that are transcribed into a single mRNA- (questions on the exam on this for sure!)

A bacterial gene unit - where several proteins are made from one single mRNA molecule. The important feature is that there is just one gene regulatory sequence on the DNA controlling the transcription of the DNA 08_06_single.promot.jpg 08_06_single.promot.jpg

08_07_repress.protein.jpg Can I get to that DNA please?

The Lac Operon The best studied operon Classic example that every cell biology student should memorize forever!!! Read all about it on page 275 - please

Eukaryotic gene expression More complicated compared to bacteria 4 steps:- 1) RNA Polymerase Bacteria have JUST one RNA polymerase Eukaryotes possess three RNA polymerases (RNA Pol II transcribes the vast majority of genes) 2) General Transcription Factors (proteins) must first assemble on the DNA before RNA Pol can attach

Eukaryotic gene expression.. 3) Eukaryotic regulators can act over vast distances from the site of gene transcription. Whilst such bacterial elements act locally 4) Complex eukaryotic DNA folding and packing has an impact on transcription too.

RNA Pol II initiation factors TFIID binds to the ‘TATA’ box - a short region of DNA located about 25 bases upstream of the gene start site TFIIA and TFIIB bind to TFIID causing local unraveling of the DNA TFIIE, TFIIH, TFIIF, and RNA Pol II bind next Addition of phosphate groups to the RNA Pol II allows transcription to commence, and results in the release of the all the other transcription initiation factors…

Learn this please 08_10_transcr.factors.jpg 08_10_transcr.factors.jpg

Mediator proteins & bendy DNA Eukaryotic genes may be regulated by other master switches which are vast distances away from the local promoters. These are known as ‘enhancer’ sequences. The cell can bring such regions together by simply ‘looping out’ the DNA in between. The enhancer and promoter are then held together by mediator proteins, which stabilize the transcription initiation complex

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Chromatin Structure and transcription Not too much is understood about the interactions between gene expression and chromatin structure We do know that heterochomatin regions of DNA do not permit gene expression due to the tight folding of the DNA around nucleosomes Histone modifying proteins - those that add acetyl groups to specific lysines in the tails of histone proteins increase access, while those that reduce acetylation result in repression of transcription

08_14_chromatin.struc.jpg 08_14_chromatin.struc.jpg