Exam 1! ! Mean = 66! Median = 66! Range (26 94)! ! A B C D F > 84 (21)! 74 to 82 (34)! 64 to 72 (45)! 50 to 62 (56)! < 48 (25)!
Exam 1 Grade Distribution!
60! 50! 40! 30! 20! 10! 0! A! B! C! D! F!
Essential Cell Biology
Third Edition
Chapter 7 From DNA to Protein: How Cells Read the Genome !
Copyright Garland Science 2010
Charles Robert Darwin !
Alfred Russell Wallace !
Francis Harry C. Crick!
James Dewey Watson!
The Central Dogma of Molecular Biology !
! Occurs in all cells from bacteria to humans.! ! ! One of the dening characteristics of living cells.! ! Allows massive amplication of signals from a single gene.!
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Caught in the Act!
Actively Transcribing Vertebrate DNA!
Figure 7-8 Essential Cell Biology ( Garland Science 2010)
The efciency of gene expression is quite variable !
! Translation efciency, as well as RNA and protein stability vary greatly among genes!
Structure of RNA !
Differs from DNA in 2 signicant ways:! ! 1. ribonucleotides vs deoxyribnucleotides! ! 2. uracil vs thymine!
RNA is typically single stranded !
! Because RNA is single stranded, intramolecular base pairing can occur, resulting in elaborate secondary structure! ! This gives rise to diverse functionality (e.g., ribozymes, riboswitches, tRNA, rRNA, )!
RNA serves many functions !
Gene expression refers to the biosynthesis of either DNA-encoded protein, or non-coding RNA !
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nal product = RNA molecules !
From an evolutionary perspective, RNA may have been the original, self-replicating biopolymer! ! ! Ribozymes may have developed the ability to direct protein synthesis! ! ! DNA is probably a relative newcomer, usurping RNAs role in information storage!
Transcription is the DNA-directed biosynthesis of a single, complementary RNA molecule !
! ! ! ! RNA is much shorter than DNA.! RNA polymerase carries out transcription.! RNA polymerase does not need a primer.! Many RNA polymerases can transcribe a single gene at the same time.!
! Transcription does have similarities to replication.! ! ! As for DNA, RNA is synthesized in the 5 to 3 direction.!
RNA polymerase = 10 subunit protein complex!
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Figure 7-7 Essential Cell Biology ( Garland Science 2010)
Eukaryotic transcription differs from bacterial transcription in a few ways !
1.! Bacteria have only 1 RNA pol. Eukaryotes have 3.!
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2.! Eukaryotic RNA polymerases require a bunch of accessory proteins called the general transcription factors (GTFs) to initiate transcription.! 3.! Control mechanisms are more complex in eukaryotes in part because genes are much further apart. This allows more sophisticated gene regulation.! 4.! Eukaryotic transcription has to deal with more compact chromatin structure.!
Bacterial promoters and terminators have specic sequences recognized by RNA polymerase !
! The promoter orients RNA pol and tells it where to start and which way to go.! ! All bacterial genes have promoter and terminator sequences similar to those shown below.!
Certain sequences of DNA tell RNA polymerase where to start (promoters) and stop (terminator/stop site) !
! ! In bacteria, the sigma factor, a subunit of the RNA pol, recognizes the promoter. ! ! This is a dynamic process.!
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In bacteria !
Either strand of DNA can act as a template, but the promoter is asymmetrical !
The General Transcription Factors !
! ! ! ! Assemble on the promoter.! Position RNA polymerase.! Open DNA.! Launch the RNA polymerase.!
typically ~25 bp upstream of start site; most promoters have this ! Both opens DNA and phosphorylates and releases RNA pol from initiation complex !
TBP is a subunit of TFIID that distorts DNA, forming landmark !
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transcription initiation complex !
! All components are then later recycled to be used again and again!
TATA Box Binding Protein distorts the double helix!
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Figure 7-13 Essential Cell Biology ( Garland Science 2010)
~ 25 bp upstream from transcription start site!
TBP distorts DNA!
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TFIIB provides scaffold!
TFIIH separates strands!
Figure 7-12 (part 1 of 2) Essential Cell Biology ( Garland Science 2010)
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TFIIF phosphorylates tail ! domain of RNA pol II ! ! ! ! ! ! . launches polymerase!
Figure 7-12 (part 2 of 2) Essential Cell Biology ( Garland Science 2010)
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Control of transcription initiation is the most common way organisms control gene expression. !
Eukaryotic RNAs must be processed and exported to cytoplasm!
! In eukaryotes, transcription occurs in nucleus, translation occurs in cytoplasm. The export of RNA occurs via nuclear pore complexes in the nuclear envelope.! ! ! Prior to nuclear export, RNA processing occurs as the RNA molecule is being synthesized.! !
RNA processing !
! Occurs as RNA is being made.! ! Processing machinery is recruited to the phosphorylated tail ! !domain of the eukaryotic RNA polymerase.! ! Different types of processing occurs depending of what type of RNA is being made. ! !
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Eukaryotic mRNA processing !
! Addition of 5-caps and 3polyadenylation tails (poly-A tails) (also intron splicing) .! ! 5-caps and poly-A tails:!
1.! Increase stability.! 2.! Identies the molecule as mRNA.! 3.! Marks the mRNA as being complete.!
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Usually gets trimmed back rst before few hundred A added. !
! !Eukaryotic genes are often interrupted by noncoding sequences (introns). Need to remove/splice these introns out to get nished/meaningful message.! ! Exons-expressed sequences! ! Introns-intervening/nonexpressed sequences!
Introns in eukaryotic mRNA are removed by RNA splicing !
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Splicing can happen in prokaryotes, but rarely does.!
Introns are removed by RNA splicing !
! ! ! Occurs during transcription after 5capping. Can occur before during or after addition of poly-A tail.! Involves cutting out introns and stitching exons back together.! Unlike exons, most of intron sequence appears to be unimportant. There are a few short sequences at or near each intron end that act as cues for removal.!
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carried our primarily by catalytic RNA molecules (small nuclear RNAs; snRNAs) coupled to a few proteins to form small nuclear ribonucleoprotein particles (snRNPs)forming the core of the Spliceosome.!
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This process carried our primarily by catalytic RNA molecules (small nuclear RNAs; snRNAs) coupled to a few proteins to form small nuclear ribonucleoprotein particles (snRNPs)forming the core of the Spliceosome.!
snRNPs bind to specic sequences at both ends of the intron.! ! The 2 hydroxyl of a conserved A attacks 5 splice site forming lariat! ! 3 hydroxyl of rst exon attacks 3 splice site, knitting exons together! ! Lariat is degraded!
Figure 7-20 Essential Cell Biology ( Garland Science 2010)
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Introns are removed by RNA splicing !
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! Alternative splicing leads to greater protein diversity from single gene.! ! ~60% of human genes undergo alternative splicing. ! ! Could have helped speed evolution of eukaryotes ! !(e.g., domain swapping ).!
The Nuclear Pore Complex recognizes and exports only completed mRNAs !
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! RNA binding proteins interact with and signal properly made mRNAs (cap binding complex, exon junction complex, polyA BP).! ! RNAs that dont pass QC are degraded and recycled.! ! Eventually all mRNA molecules are degraded into nucleotides via RNases. Sequences in the 3-UTR help determine stability.!
mRNA processing summary !
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Chicken & Egg question! ! Transcript lifetimes vary! (~ 3 min for procaryotes, ~ 30 min for eucaryotes)!
Translation is the RNA-directed biosynthesis of proteins !
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In order to unambiguously specify 20 amino acids using a code composed 4 letters (bases), need at least 3 letters = codon! ! There are 43 = 64 permutations = unique codons!
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Thus, the genetic code is redundant some AAs specied by more than one codon.! ! Punctuation marks = AUG (start; also Met); UAA, UAG, UGA = stop!
RNA can be read in any of 3 open reading frames !
Appropriate ORF is signaled by the translation start site. ! !
Codons specify binding sites for tRNAs !
! tRNA = transfer RNA! ! Only 31 different tRNAs for 64 codons.! ! Some tRNAs recognize > 1 codon (wobble phenomenon).!
D = dihydrouridine; ! = pseudouridine"
Correct charging of tRNAs requires enzymes called aminoacyl-tRNA synthetases !
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! Each AA is charged to one or more UNIQUE t-RNAs.!
Ribosomes are enormous proteinmanufacturing machines !
! Ribosomes are made of > 80 different proteins (ribosomal proteins) and 4 RNA molecules (rRNAs). ! ! Composed of 1 large and 1 small subunit.! ! Highly conserved.!
Ribosomes are elaborate ribozymes !
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~ 2:1 RNA:Protein !
catalyzes formation of the peptide bonds!
matches tRNAs to mRNA codons!
Subunits separate after protein synthesis is completed !
Three t-RNA binding sites on ribosome!
Figure 7-32 Essential Cell Biology ( Garland Science 2010)
Each ribosome has 1 binding site for the mRNA and 3 binding sites for the tRNAs!
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Protein grows from N- to C-terminus !
Figure 7-33 (part 1 of 5) Essential Cell Biology ( Garland Science 2010)
Figure 7-33 (part 2 of 5) Essential Cell Biology ( Garland Science 2010)
Figure 7-33 (part 3 of 5) Essential Cell Biology ( Garland Science 2010)
Figure 7-33 (part 4 of 5) Essential Cell Biology ( Garland Science 2010)
Codons within the mRNA signal where to start and stop translation !
! Translation begins with AUG start codon.!
cap recognized!
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! Need special initiator tRNA and translation initiation factors.! ! Special initiator tRNA is used at the start codon that always carries methionine. Only tRNA that can bind straight to the P site.!
! This methionine is usually removed later by a specic protease.!
! Bacteria use a Shine-Delgarno sequence (~6 bps long) upstream of the AUG to cue the start of translation, while eukaryotes use a Kozak sequences.!
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+EF-Tu!
Prokaryotic mRNA is usually polycistronic !
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! Prokaryotic ribosomes can recognize and bind near start codons in the middle of an mRNA molecule.! ! ! Several related proteins are synthesized simultaneously from a single mRNA!
Translation is terminated at stop codons !
! UAA, UAG and UGA! ! They signal the binding of release factors.! ! ! A hydrolysis reaction then frees the polypeptide.! ! Nascent proteins are typically met by chaperone proteins as they emerge from the ribosome!
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Proteins are made on polyribosomes (polysomes) !
! During efcient protein synthesis, a new ribosome hops onto the mRNA right after the preceding ribosome moves out of the way.!
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Posttranslational modications !
! After biosynthesis, many proteins undergo posttranslational modications that can substantially alter protein stability and function.!
Many antibiotics target prokaryotic translation !
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! Weve been able to borrow much of this technology from our eukaryotic cousins, the fungi.!
Proteins can have vastly different life spans !
! Proteins are degraded by proteases.! ! Many proteins are degraded by proteosomes. Requires ubiquitin tags. ! ! However, other mechanisms like autophagy also occur.! proteosome ! autophagy !
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Gene expression summary !
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End!
