Molecular Biotechnology Lecture 2: Genes, Genome Structure, and Inheritance - Prof. Jennif, Study notes of Bioinformatics

Download Molecular Biotechnology Lecture 2: Genes, Genome Structure, and Inheritance - Prof. Jennif and more Study notes Bioinformatics in PDF only on Docsity! BINF 6010/8010 Fall 2008 Molecular Biotechnology hLecture 2: Aug 26t , 2008 Lecturer: Dr. Weller Course Web pages: http://webpages.uncc.edu/~jweller2 Lecture 2: Agenda • Inheritance and Information content of the genome • DNA, RNA and protein synthesis – molecular biology 8/28/2008 Weller BINF6010/8010 BG@UNCC 2 Genome Structure • The structure of a genome is the information organization of the genes and regulatory regions along a contiguous piece of DNA I f ti P ki– n orma on ac ng • Operons, gene density, overlapping genes, coding strand use • The physical organization in space also imposes constraints. Physical Packing– • Shape/integrity: circular versus linear • Chromatin and higher orders: looping, etc 8/28/2008 Weller BINF6010/8010 BG@UNCC 5 Data Information • Information is provided by genomics and bioinformatics – they provide the mapping of the parts to one another and to the genome. – Primary DNA sequence (collection, integration, QC) – Assignment of sequence to physical units (plasmids, chromosomes, etc) – Annotation of sequence with regulatory versus coding sequence information (note that coding might mean protein-coding or RNA- gene coding) • What to expect from the next generation comes from – Inheritance rules and exceptions – DNA replication and fidelity rules 8/28/2008 Weller BINF6010/8010 BG@UNCC 6 Affecting Inheritance • Copies of genomic DNA for offspring are produced via DNA replication – What is the error rate for the genome? • The replication error rate is less than 1 in one billion (3 x 10 -10 or one per genome for humans) for chromosomal DNA in eukaryotes and many prokaryotes. • BUT some genetic diseases in humans are due to a mutation in a nuclear- coded polymerase that leads to 100-fold higher mtDNA frameshift mutations i.e. 6 x 10-8 per mitochondrial genome replication – Is the error rate consistent and is it completely random? i.e. are there hotspots, are ‘typical’ errors clustered in certain regions? • The fragile X region is subject to amplification between generations – What type of recombination occurs? – How many alleles are common for each gene in the study population – How many pseudogenes are there? Are they expressed as transcripts? 8/28/2008 Weller BINF6010/8010 BG@UNCC 7 DNA review • DNA and RNA are chemically similar polymers – The four types of monomer subunits differ in the aromatic base attached to the sugar (A,T or U,C,G) and the sugar (with or without the 2’ hydroxyl) – Sugar-phosphate backbone – The monomers differ in the nature of the aromatic compound (the base) attached to the sugar. • The monomers are not symmetrical, so the backbone connecting the monomers has polarity – the chain has a direction which we define as (5’ -> 3’ is the notation convention, shown next). – This means that the order of the bases is significant (otherwise TGC and CGT would be equivalent isomers, with much less information content) 8/28/2008 Weller BINF6010/8010 BG@UNCC . 10 Monomers 8/28/2008 Weller BINF6010/8010 BG@UNCC Images from Lodish et al, on-line at ncbi 11 Bases 8/28/2008 Weller BINF6010/8010 BG@UNCC Images from Lodish et al, on-line at ncbi 12 8/28/2008 Double helix Normal B DNA Weller BINF6010/8010 BG@UNCC 15 A SUMMARY OF DNA REPLICATION Single-strand binding The leading strand is proteins stabilize the synthesized continuously unwound parental DNA. in the 5°—> 3’ direction by DNA polymerase. @ helicases unwind the DNA polymerase parental double helix. The lagging strand is synthesized discontinuously. RNA primer Primase synthesizes a short Okazaki fragment RNA primer, which Is extended by DNA polymerase 5 being made to form an Okazaki fragment. - DNA Parental DNA P0Olymerase © Atter the ANA primer is replaced by DONA (by another DNA polymerase, not shown), DNA ligase joins the Okazaki fragment to the growing DNA ligase strand. cc —_:::_._c_cccCcCc_‘“ Overall direction of replication http:/Awww.esb.utexas.edu/mabrybio2 11/chapter05/74-S ummary-DNA-Replication BG@UNCC DNA mRNA • Transcription of DNA to RNA resembles DNA replication: a unidirectional chemical reaction mediated by enzymes, tuned by chemical effectors. – The RNA polymerase holoenzyme performs the activity • a supra-molecular complex of many different protein factors • directs the synthesis of mRNA on a DNA template • Requires a template, NTP monomers, energy, Mg++ – Requires information signals from the DNA template – regulatory regions – where to stop, start, how much, etc. 8/28/2008 Weller BINF6010/8010 BG@UNCC 17 Regulatory Sequences • The core replication and transcription proteins recognize DNA sequences and bind to them – Other factors bind to those proteins and modulate them • Change the rate change the sensitivity to other factorsr , • Signals include promoter elements, enhancers, transcription terminators • Secondary signals include intervening sequences, base modification signals, etc. • A great deal of molecular biotechnology involves not just the alteration of the gene coding sequence (the triplet codons) but of non-coding sequences that govern how and when the mRNA is made and how stable it is. 8/28/2008 Weller BINF6010/8010 BG@UNCC 20 Promoter Elements • RNA polymerase must know where to start in the genome: recognize the beginning of a gene . – There is a DNA sequence at the beginning of genes for which one of the polymerase subunits has high affinity. – It is unidirectional on one strand so it is both start signal and direction is gn. • The bacterial promoter almost always contains some version of the following elements: 8/28/2008 Weller BINF6010/8010 BG@UNCC 21 Termination of Transcription • Stopping in Prokaryotes: At the end of a gene, the sequence of the mRNA allows it to form a hairpin loop, which blocks the ribosome. The ribosome falls off the mRNA and as soon as the ribosome– , falls off the mRNA, the RNA polymerase falls off the DNA and transcription ceases. St i i E k t A l d l ti i l i• opp ng n u aryo es: po y-a eny a on s gna s recognized by factors that cleave the RNA, so AAA is added to the 3’ tail; – the RNA plI keeps going but with some factors now gone from the holoenzyme, at which point a ribonuclease binds and degrades (5’-3; exonuclease) until it catches up with RNApII, 8/28/2008 Weller BINF6010/8010 BG@UNCC when the complext detaches. 22 Polycistronic mRNA All of the genes are made as a single transcript, and ribosomes read through the transcript to make a large polypeptide that is cleaved post-translationally 8/28/2008 Weller BINF6010/8010 BG@UNCC 25 to get the individual proteins. www.nitro.biosci.arizona.edu/courses/EEB600A-200 Eukaryotic mRNA processing • In eukaryotes the primary mRNA transcript undergoes many processing steps. – The primary transcript has • untranslated regions (UTRs at both the 5’ and 3’ termini) that are important for regulation and correct processing • intergenic sequences (introns) between the segments that code for protein (exons) and must be removed by the splicing apparatus. • Signals for addition of polyA • The mature message has – A ‘cap’ structure, a modified G nucleotide is added to the 5’ end A l A t il i dd d t th 3’ d (thi i d th t bilit– po y a s a e o e en s ncrease e s a y, or half-life, of the mRNA) – The introns are spliced out 8/28/2008 Weller BINF6010/8010 BG@UNCC 26 Introns and Exons Ovalbumin gene, 7700 bp eae 123456 7 Ovalbumin mRNA — 1872 nucleotides ———>| ‘Copyright 1989 John Wiley and Sons, inc. All rights resarved. http:/Avwww.mun.ca/biochem/courses/3107/images/VVP/Ch25/25-18.jpg 27 BG@UNCC Utranslated Regions: UTRs • UTRs contain a lot of regulatory information for the transcript. 5’ start stop 3’ codon codon | |------------- -------------------------------------------- ------------ 5'-UTR translated RNA 3'-UTR • In the 3’ UTR are elements that – Signal polyadenylation – Signal for modified amino acid selenocysteine (SECIS elements, direct the ribosome to translate UGA codons as selenocysteines). The histone downstream element– 8/28/2008 Weller BINF6010/8010 BG@UNCC 30 Splice variants • Splice variants: Alternative splicing allows exons from the same nuclear mRNA to be combined in different ways to give different proteins. – Alternative splicing often occurs when a protein specific for a type of cell is made. Th t th b d j ti b t– e sequence a e oun ary- unc ons e ween introns and exons provide the information about the possibility for forming splice forms. • Bioinformatics tools that can predict splice variants include Gene Finder and HMMgene. 8/28/2008 Weller BINF6010/8010 BG@UNCC 31 Splice Forms Cartoon Exon Exon Exon Exon Gene mRNA [1] [2] [3 eae Alternative Splicing iia aaa Protein A Protein B mRNA bttodAMvaww.ncbi.nim.nih.gov/Class/MLACoUtse/Modules/MolBioReview/images/alter native splicing. gif BG@UNCC Protein Synthesis Eukaryotes • The mRNA must be processed and t t d t th t l i t t l tiranspor e o e cy oso pr or o rans a on • Eukaryotes: translation initiation starts with the assembly of a complex on the 5′ cap that i l d th 40S b it d M t tRNAnc u es e su un an e - i. • Requires energy (ATP hydrolysis) which a helicase performs • The complex scans the mRNA until the first AUG is reached. • Then the 60S subunit is then added to form the 80S initiation complex. • There are a number of protein helpers for both the initiation and elongation phases, and one for the termination step. 8/28/2008 Weller BINF6010/8010 BG@UNCC 35 Directionality of biological polymers (regulatory region 5’) 3 kbp of DNA start stop RNA polymerase 5’ 3’ transcription only goes one way N C start stop Protein synthesis only goes one way In prokaryotes there is a 1:1 correspondence from DNA to RNA; in eukaryotes there may be interruptions, caused by introns, alternative li i l 8/28/2008 Weller BINF6010/8010 BG@UNCC sp ce s gna s, etc 36