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Today… Genome 351, 12 April 2013, Lecture 4 •mRNA splicing •Promoter recognition •Transcriptional regulation •Mitosis: how the genetic material is partitioned during cell division In bacteria (most) mRNAs are co-linear with their corresponding genes Promoter terminator gene AACTGACGA +1 bacteria: AACUGACGA AACGA mRNA Events involved in RNA processing Pre-mRNA Intron Exon1 Noncoding Coding sequence Noncoding Exon2 Coding sequence Noncoding Noncoding Continuous stretch of coding sequence Noncoding Noncoding Continuous stretch of coding sequence Noncoding Transport to the cytoplasm AAAAA Why does transcript splicing occur? Proteins can be modular -Different regions can have distinct functions and the modules can correspond to exons Interrupted structure allows genes to be modular secretion cell anchor enzyme binding module Interrupted structure allows genes to be modular Pre-mRNA: secretion cell anchor enzyme binding module secretion cell anchor enzyme binding module secretion cell anchor enzyme binding module Processed-mRNA AAAA Alternative splicing or: One mRNAs exon is another one’s intron! Pre-mRNA: secretion secretion cell anchor enzyme enzyme binding module binding module one alternative form secretion enzyme binding module AAAA Processed-mRNA Alternative splicing or: One mRNAs exon is another one’s intron! Pre-mRNA: secretion enzyme enzyme cell anchor binding module binding module another alternative form enzyme binding module AAAA Processed-mRNA How do RNA polymerases know where to begin transcription and which way to go? promoter mRNA mRNA gene promoter gene gene promoter mRNA First worked out in bacteria by: -comparing sequences near the start sites of transcription of many genes -by studying where RNA polymerase likes to bind to DNA How do RNA polymerases know where to begin transcription and which way to go? Comparing sequences at the promoter region of many bacterial genes provides clues: direction of transcription only coding (sense) strand transcription start site is shown; all sequences 5’-3’ -35 region -10 region consensus sequence: TTGACAT…15-17bp…TATAAT +1 RNA polymerase binds to the consensus sequences in bacterial promoters RNA polymerase binds to the -35 and -10 regions: RNA polymerase direction of transcription TTGACAT -35 region TATAA T -10 region +1 Would you expect RNA polymerase to bind the other way around and transcribe in the reverse direction? RNA polymerase binds to the consensus sequences in bacterial promoters RNA polymerase binds to the -35 and -10 regions: RNA polymerase direction of transcription TTGACAT -35 region TATAA T -10 region +1 Would you expect RNA polymerase to bind the other way around and transcribe in the reverse direction? RNA polymerase binds to the consensus sequences in bacterial promoters direction of transcription RNA polymerase TTGACAT -35 region TATAA T -10 region +1 Would you expect RNA polymerase to bind this sequence and initiate transcription? T T direction of transcription AATA T ACAGTT How do RNA polymerases know where to begin transcription and which way to go? In bacteria RNA polymerase binds specific sequences near the start site of transcription that orient the polymerase: mRNA gene mRNA gene TTGACAT TATAAT -35 region -10 region gene mRNA -10 region -35 region TAATAT TACAGTT In eukaryotes, RNA polymerase is regulated by DNA-binding proteins transcription factors (TF’s): But TF’s that bind to specific DNA sequences & to RNA polymerase can recruit RNA polymerase & activate transcription RNA polymerase: +1 RNA polymerase does not efficiently bind to DNA and activate transcription on its own +1 In eukaryotes, RNA polymerase is regulated by DNA-binding proteins transcription factors (TF’s): But TF’s that bind to specific DNA sequences & to RNA polymerase can recruit RNA polymerase & activate transcription RNA polymerase: +1 Some TF’s can also inhibit transcription +1 Switches and Regulators - A Metaphor • Switches control transcription (which take the form of DNA sequence) - Called regulatory elements (RE’s) or enhancers - Adjoin the promoter region, but can be quite distant • Regulators, which take the form of proteins that bind the DNA, operate the switches - Called transcription factors (TF’s) • When and how much RNA is made often is the product of multiple elements and regulators Control of gene expression •Each cell contains the same genetic blueprint •Cell types differ in their protein content •Some genes are used in almost all cells (housekeeping genes) •Other genes are used selectively in different cell types or in response to different conditions. An imaginary regulatory region RE1 RE2 RE3 RE4 RE5 RE6 Promoter Expressing a regulatory gene in the wrong place can have disastrous consequences!!! Example: Antennapedia gene in fruit flies Antennapedia gene is normally only transcribed in the thorax; legs are made. A mutant promoter causes the Antennapedia gene to be expressed in the thorax and also in the head, where legs result instead of antennae! Lactose tolerance: A human example of a promoter mutation Lactase levels Lactase levels in humans 2 10 Age in years World wide distribution of lactose intolerance The cellular life cycle Mitosis: dividing the content of a cell fertilized egg; a single cell! Chromosomes - a reminder How many do humans have? •22 pairs of autosomes •2 sex chromosomes •Each parent contributes one chromosome to each pair •Chromosomes of the same pair are called homologs •Others are called nonhomologous Photo: David McDonald, Laboratory of Pathology of Seattle Homologous and non-homologous chromosomes The zygote receives one paternal (p) and one maternal (m) copy of each homologous chromosome 1p 2p 3p 1m 2m 3m 21p 22p Xp or Y 21m 22m Xm The DNA of human chromosomes # base pairs # genes # base pairs # genes The cellular life cycle Elements of mitosis: cell growth; chromosome duplication cell growth; chromosome duplication chromosomes decondensed chromosome segregation chromosomes condensed chromosome segregation repeat Chromosome replication – a reminder • Mechanism of DNA synthesis ensure that each double stranded DNA gets copied only once. • The products of DNA replication have one new DNA strand and one old one (semi-conservative replication) Chromosome structure – a reminder chromosome structure during cell growth & chromosome replication (decondensed) held together at the centromere sister chromatids; doublestranded DNA copies of the SAME homolog Mitosis -- making sure each daughter cell gets one copy of each pair of chromosomes • Copied chromosomes (sister chromatids) stay joined together at the centromere. • Proteins pull the two sister chromatids to opposite poles • Each daughter cell gets one copy of each homolog. Mitosis -- homologous chromosomes 1m 1p 2 copies 1m 2 copies 1p 2 copies 1m 2 copies 1p 1m 1p 1m 1p 1m 1p 1m 1p exact copies joined at centromer e Mitosis – following the fate of CFTR CFTR+ 2 copies CFTR+ 2 copies CFTR+ CFTR+ CFTRCFTR+ CFTR- CFTR2 copies CFTR2 copies CFTRCFTR+ CFTRCFTR+ CFTR- A CFTR heterozygote (CFTR+/CFTR-) A closer look at the chromosomes Paternal chromosome CTCCTCAGGAGTCAGGTGCAC GTGCACCTGACTCCTGAGGAG CTCCACAGGAGTCAGGTGCAC GTGCACCTGACTCCTGTGGAG Maternal chromosome Mitosis -- 2 copies of each chromosome at the start A closer look at the chromosomes CTCCTCAGGAGTCAGGTGCAC GTGCACCTGACTCCTGAGGAG CTCCACAGGAGTCAGGTGCAC GTGCACCTGACTCCTGTGGAG DNA strands separate followed by new strand synthesis A closer look at the chromosomes CTCCTCAGGAGTCAGGTGCAC GTGCACCTGACTCCTGAGGAG CTCCTCAGGAGTCAGGTGCAC GTGCACCTGACTCCTGAGGAG CTCCACAGGAGTCAGGTGCAC GTGCACCTGACTCCTGTGGAG CTCCACAGGAGTCAGGTGCAC GTGCACCTGACTCCTGTGGAG • Mitosis -- after replication 4 copies • Homologs unpaired sister chromatids joined by centromere A closer look at the chromosomes CTCCTCAGGAGTCAGGTGCAC GTGCACCTGACTCCTGAGGAG CTCCACAGGAGTCAGGTGCAC GTGCACCTGACTCCTGTGGAG CTCCTCAGGAGTCAGGTGCAC GTGCACCTGACTCCTGAGGAG CTCCACAGGAGTCAGGTGCAC GTGCACCTGACTCCTGTGGAG Each daughter has a copy of each homolog Mitosis and the cell cycle Mitosis vs. Meiosis - The goal of mitosis is to make more “somatic” cells: each daughter cell should have the same chromosome set as the parental cell - The goal of meiosis is to make sperm and eggs: each daughter cell should have half the number of chromosome sets as the parental cell Meiosis: the formation of gametes The challenge: • ensuring that homologues are partitioned to separate gametes The solution: • Hold homologous chromosomes together by crossing over • target homologues to opposite poles of the cell… • then separate the homologues