Introduction to Molecular Biology PDF

Introduction to Molecular Biology  Molecular Biology Study of gene structures and their functions Study of process of replication, transcription and translation of genetic material. Interaction b/w DNA, RNA and protein bio- synthesis.  Definition of Cell A cell is the smal...

Introduction to Molecular Biology  Molecular Biology Study of gene structures and their functions Study of process of replication, transcription and translation of genetic material. Interaction b/w DNA, RNA and protein bio- synthesis.  Definition of Cell A cell is the smallest unit that is capable of performing life functions.  Cell Theory All living things are made up of cells. Cells are the smallest working units of all living things. All cells come from preexisting cells through cell division.  Two Types of Cells Prokaryotic Eukaryotic  Prokaryotic Do not have structures surrounded by membranes Few internal structures One-celled organisms, Bacteria http://library.thinkquest.org/C004535/prokaryotic_cells.html  Eukaryotic Contain organelles surrounded by membranes Most living organisms Plant Animal http://library.thinkquest.org/C004535/eukaryotic_cells.html Prokaryotic and Eukaryotic cells Eukaryotes: Organisms whose cells contain compartments or organelles within the cell, such as mitochondria and nucleus – Animals, plants Prokaryotes: Whose cells do not have these organelles (e.g. bacteria) – Most prokaryotes have a smaller genome, typically contained in a single circular DNA molecule. – Additional genetic information may be contained in smaller satellite pieces of DNA called plasmids  Cells, genome, gene and DNA Almost all cells of a living organism contain an identical set of codes describing the genes and their regulation This code is encoded as one or more strands of DNA Cells from the different parts of an organism have the same DNA – Distinction: The portion of the DNA that is transcribed and translated into protein Genome: entire complement of DNA molecules of each organism Overall function of genome: Control the generation of molecules (mostly proteins) that will – Regulate the metabolism of a cell and its response to the environment, and – Provide structural integrity.  Structure of DNA Made up of 4 different building blocks (so called nucleotide bases), each an almost planar nitrogenic organic compound – Adenine (A) – Thymine (T) – Guanine (G) – Cytosine (C) – Base pairs (A -- T, C -- G)  DNA function Carries the blueprint for life Duplication for new cells Make proteins for biological functions:  Structure of DNA -- Base pairs (A -- T,C -- G) are attached to a sugar phosphate backbone to form one of 2 strands of a DNA molecule. – Phosphate ((PO4) -3) – Deoxyribose Two strands are bonded together by the base pairs (A – T, C – G). Results in mirror image or complementary strands, each is twisted (or helical), and when bonded they form a double helix. Direction of each strand (5’ meaning beginning or 3’ meaning end of the strand) – 5’ and 3’ refer to position of bases in relation to the sugar molecule in the DNA backbone. – Are important reference points to navigate the genome. – 2 complementary strands are oriented in opposite direction to each other. Structure of DNA --  Duplication of DNA Occurs through the coordinated action of many molecules, including – DNA polymerases (synthesizing new DNA), – DNA gyrases (unwinding the molecule), and – DNA ligases (concatenating segments together)  Transcription of DNA to RNA Why transcription: – (For genome) to direct or effect changes in the cytoplasm of the cell – Need to generate new proteins to populate the cytosol (heteregenous intracellular soup of the cytoplasm) Note: DNA is in the nucleus, while proteins are needed in the cytoplasm, where many of the cell’s functions are performed. Coding region of the DNA is copied to a more transient molecule called RNA – Gene is a single segment of the coding region that is transcribed into RNA – Generation of RNA from DNA (in the nucleus) is done trough a process called transcription  Transcription RNA (Ribonucleic acid) – Similar to DNA (except for a chemical modification of the sugar backbone) – Instead of T contains U (Uracil) which binds with A. – Is not double stranded but single stranded – RNA molecules tend to fold back on themselves to make helical twisted and rigid segments. RNA is synthesized – By unwinding the DNA double helix separating the 2 strands. – Using one of the strands as a template along which to build the RNA molecule – Accomplished by Enzyme RNA polymerase (binds to promoter and copies or transcribes the gene in its full length) – Resulting molecule is called Pre-mRNA – Single stranded pre-mRNA is then processed. – Splicing (mediated by spliceosome consisting of RNA and proteins) removes the introns. – Ends modified (Capping modifies 5’ end and Polyadenylation adds adenines at the 3’ end) to enhance stability  mRNA, ORFs, etc. Each cell has 20 to 30 pg of RNA (1% of the cell mass) The RNA that codes for proteins is called messenger RNA (mRNA) The part of DNA that provides that code is called Open Reading Frame (ORF) When read in the standard 5’ to 3’ direction, the portion of DNA before the ORF is considered upstream and the portion following the ORF is considered downstream. Promoter regions: DNA sequence upstream of an ORF – Specifically determine which gene to transcribe – Transcription factors: proteins that contain part that bind to specific promoter regions, thus activating or deactivating transcription of the downstream ORF  Coding and non-coding RNA Not all RNA code for proteins – 4% of total RNA is made of coding RNA – Of the non-coding RNA Ribosonal RNA (rRNA) and transfer RNA(tRNA) are used in the various protein translational apparatus Small nuclear RNA (snRNA) – found in eucaryotes, is part of the splicing apparatus Small nucleolar RNA (snoRNA) involved in methylation of rRNA Small cytoplasmic RNA (scRNA) plays a role in the expression of specific genes  More on transcription Most eukaryotic genes have exons (portions that will be put in the mRNA) and introns (that are normally spliced out) – Some introns may have a promoter-like control of the transcription process – If an intron is not spliced out then an alternative splicing product is created. – Various tissue types can flexibly alter their gene products through alternative splicing Post-splicing (in Eukaryotes) – The generated mRNA is exported (through nuclear pore complexes) to the cytoplasm – In the cytoplasm, the ribosonal complex (containing hundreds of proteins and special function RNA molecules) acts to generate the protein on the basis of the mRNA code.  Translation Process of generating a protein or polypeptide from an mRNA molecule is known as translation. Protein: a polymer or chain of aminoacids, whose sequence is determined by the mRNA template – 3 nulceotides code for 20 naturally occurring amino acids – 43 = 64; thus several trinucleotide sequences (codons) correspond to a single amino acid. – There is no nucleotide between codons, and a few codons represent start and stop. – Notable exceptions: code of naturally occurring selenocysteine is identical to that for a stop codon, except for a particular nucleotide sequence further downstream.  Proteins Huge proportion of cell (after water) Many functions: – Structure (e.g. collagen in bone) – Enzymes – Transmembrane receptors – Hormones Four levels of structure Protein structure  Protein interactions Proteins can form interations: – Proteins (complexes, oligomers) – mRNA – DNA Proteins can bind to each other depending on their relative charges and structures  Translocation of proteins A newly formed protein need to be translocated to the right place to perform its function (such as structural protein in the cytoskeleton, as a cell membrane receptor, as a hormone that is to be secreted by the cell, etc.) Signal peptide (header): part of the polypeptide that is one of the determinant of its location and handling Gene expression regulation  Transcriptional programs Initiation of the transcription process can be caused by external events or by a programmed event within the cell. External events – Piezoelectric forces generated in bones through walking can gradually stimulate osteoblastic and osteoclastic transcriptional activity to cause bone remodelling; Heat shock – Appearance or disappearance of new micro or macronutrients around the cell; binding of distantly secreted hormones Internally programmed sequences of transcriptional expression (eg. clock and per genes) Pathological internal derangements of the cell – Self-repair or damage detection programs can trigger apoptosis (self-destruction) under conditions such as irreparable DNA damage  Biological function of proteins Enzyme catalysis: DNA polymerases, lactate dehydrogenase, trypsin Transport: hemoglobin, membrane transporters, serum albumin Storage: ovalbumin, egg-white protein, ferritin Motion: myosin, actin, tubulin, flagellar proteins Structural and mechanical support: collagen, elastin, keratin, viral coat proteins Defense: antibodies, complement factors, blood clotting factors, protease inhibitors Signal transduction: receptors, ion channels, rhodopsin, G proteins, signalling cascade proteins Control of growth, differentiation and metabolism: repressor proteins, growth factors, cytokines, bone morphogenic proteins, peptide hormones, cell adhesion proteins Toxins: snake venoms, cholera toxin  Gene expression studies Allow you to understand how a gene is regulated in a tissue or a cell type. Most useful way of studying gene expression is by measuring the levels of mRNA produced from a particular gene in a particular tissue. Application: to understand certain biological process it is useful to study the differences in gene expression which occur during such processes. E.g. – It is of interest to know which genes are induced or repressed, say in the liver, after a particular drug is taken. – Or which genes are expressed in a tumor but not in the surrounding normal tissue. Some techniques for analyzing mRNA level of a single gene or to quantify gene expression – Northern blots – Quantitative reverse transcriptase PCR (QT-RT-PCR) – DNA microarrays – Proteomics (analysis of the protein synthesis that results from gene expression)  SNPs (single nucleotide polymorphisms) Genetic basis for organismal diversity is due in large part to differences in sequences, also known as polymorphisms of each gene. Most of these polymorphisms differ from one another by one nucleotide and are known as SNPs. Due to the small portion of the genome coding for proteins and the redundancy in the mRNA code, only some SNPs will result in differently constructed proteins. It is believed that genomic markers such as SNPs spaced every 1000 bases will be sufficient to unambiguously resolve the span of genome associated with a phenotypic difference to a single gene.  Gene clustering dogma Genes that appeared to be expressed in similar patterns are mechanistically related. I.e., if we can find genes whose expression patterns approximate one another we can possibly conclude that they have functions that are related.  Common terminology Genome/proteome Genotype/phenotype Pseudogene Novel protein/gene Putative gene Locus/Allele/Chromosome region Dominant/Recessive Homologous Symbol cDNA Motif Dalton (Da or kDa)  Future of molecular biology Personalised medicine Target-specific drugs (e.g. adipose tissue) Gene therapy Comparative genomics