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Review from last time. Coding and non-coding repeats make up the moderately repetitive portion of the eukaryotic genome Polyploidization and gene duplication contribute to structural and functional genome change Transposable elements contribute to structural and functional genome change

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review from last time
Review from last time
  • Coding and non-coding repeats make up the moderately repetitive portion of the eukaryotic genome
  • Polyploidization and gene duplication contribute to structural and functional genome change
  • Transposable elements contribute to structural and functional genome change
  • Comparative genomics is a useful technique to determine the conserved (likely functional) portions of a genome
this chapter in one slide
This Chapter in One Slide

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gene expression
Gene Expression
  • RNA – Ribonucleic acid
    • Slightly different from DNA
    • Uracil instead of Thymine
  • RNA is critical to all gene expression
  • mRNA – messenger RNA; created from a DNA template during transcription
  • tRNA – transfer RNA; carriers of amino acids; utilized during translation
  • rRNA – ribosomal RNA; the site of translation
  • Other RNAs – snoRNA, snRNA, miRNA, siRNA
  • Many RNAs fold into complex secondary structures
transcription
Transcription
  • Transcription – the process of copying a DNA template into an RNA strand
  • Accomplished via DNA dependent RNA polymerase (aka RNA polymerase)
transcription7
Transcription
  • By the end of this series, you should be able to explain much of this animation
  • http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf
slide8

Bacterial RNA polymerase 50 - 100 nucleotides/sec

  • Most genes are transcribed simultaneously by numerous polymerases
  • Polymerase moves along DNA in 3' —> 5' direction
  • Complementary RNA constructed in ____ direction
    • RNAn + NPPP —> RNAn+1 + PPi
transcription9
Transcription
  • How does the polymerase know where to start?
    • Promoter = the assembly point for the transcription complex
  • RNA polymerases cannot recognize promoters on their own - transcription factors
    • Transcription factors - enzymes have evolved to recognize (physically interact with) specific DNA sequences and with other proteins
    • The promoter is one such DNA sequence
transcription10
Transcription
  • Prokaryotic Transcription
  • Similar DNA sequences are seen associated with genes in roughly the same location for multiple genes in bacteria
    • The consensus sequence is the most common version of such a conserved DNA sequence
    • DNA sequences just upstream from a large number of bacterial genes have 2 short stretches of DNA that are similar from one gene to another (-35 region & -10 region)
      • T78T82G68A58C52A54 -- 162117521819 -- T82A89T52A59A49T89
      • - 35 region spacer -10 region
  • TTGATA
  • TTGACA
  • CTGACG
transcription11
Transcription
  • Prokaryotic Transcription
  • Bacterial promoters are located just upstream of the RNA synthesis initiation site
    • The nucleotide at which transcription is initiated is called +1; the preceding nucleotide is –1
    • DNA preceding initiation site (toward template 3' end) are said to be upstream
    • DNA succeeding initiation site (toward template 5' end) are said to be downstream
transcription12
Transcription
  • Prokaryotic Transcription
  • One RNA polymerase with 5 subunits tightly associated to form core enzyme
  • Core enzyme minus sigma (σ) factor will bind to any DNA.
    • By adding σ, RNA pol will bind specifically to promoters (-10 & -35 sequences)
transcription13
Transcription
  • Eukaryotic vs. Prokaryotic Transcription
  • Much of what we know is derived from studies of RNA pol II from yeast
    • 1. Seven more subunits than its bacterial RNA pol
    • 2. The core structure & the basic mechanism of transcription are virtually identical
    • 3. Additional subunits of eukaryotic polymerases are thought to play roles in the interaction with other proteins
    • 4. Eukaryotes require a large variety of accessory proteins or transcription factors (TFs)
review from last time14
Review from last time
  • Basic ideas behind transcription and translation
  • To get from DNA to functional protein, many types of RNA are critical
  • RNA differs chemically from DNA in only two ways
  • RNA tends to form secondary structures
  • RNA polymerase initiates transcription at promoter sites with the aid of transcription factors like sigma (in prokaryotes)
  • Promoters are DNA sequences that act to direct RNA polymerases to the appropriate position
transcription15
Transcription
  • Eukaryotic Transcription - one major difference
  • Three distinct RNA polymerases, each responsible for synthesizing a different group of RNAs
    • RNA polymerase I (RNA pol I) - synthesizes the larger rRNAs (28S, 18S, 5.8S)
    • RNA polymerase II (RNA pol II)- synthesizes mRNAs & most small nuclear RNAs (snRNAs & snoRNAs)
    • RNA polymerase III (RNA pol III) - synthesizes various small RNAs (tRNAs, 5S rRNA & U6 snRNA)
transcription16
Transcription
  • Eukaryotic Transcription - RNA processing
  • All major RNA types (mRNA, tRNA, rRNA) must be processed
    • Terminology
    • The primary (1°) transcript is equivalent in length to the DNA transcribed
    • The corresponding segment of DNA from which 1° transcript is transcribed is called transcription unit
    • The 1° transcript is short-lived; it is processed into smaller, functional RNAs
    • Processing requires variety of small RNAs (90 – 300 nucleotides long) & their associated proteins
transcription mrna
Transcription – mRNA
  • Messenger RNAs (mRNA)
  • Transcribed by RNA pol II
  • Remember this?
  • http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf
  • Polymerase II promoters lie to 5' side of each transcription unit
    • In most cases, between 24 & 32 bases upstream from transcription initiation site is a critical site
    • Consensus sequence that is either identical or very similar to 5'-TATAAA-3‘, the TATA box
    • The site of assembly of a preinitiation complex
      • contains the GTFs & the polymerase
      • must assemble before transcription can be initiated
transcription mrna18
Transcription – mRNA
  • The preinitiation complex
  • Step 1 - binding of TATA-binding protein (TBP)
    • Purified eukaryotic polymerase, cannot recognize a promoter directly & cannot initiate accurate transcription on its own
    • TBP is part of a much larger protein complex called TFIID
    • TBP kinks DNA and unwinds ~1/3 turn
transcription mrna19
Transcription – mRNA
  • The preinitiation complex
  • Step 2 – Binding of ~8 TAFs (TBP- associated factors) to make up the complete TFIID complex
  • Step 3 – Binding of TFIIA (stabilizes TBP-DNA interaction) and TFIIB (involved in recruiting other TFs and RNA pol II)
transcription mrna20
Transcription – mRNA
  • The preinitiation complex
  • Step 4 – RNA pol II and TFIIF bind via recruitment by TFIIB
  • Step 5 – TFIIE and TFIIH bind
  • TFIIH is the key to activating transcription in most cases
  • TFIIH is a protein kinase – phosphorylates proteins
  • TFIIH may also act as a helicase
transcription mrna21
Transcription – mRNA
  • The preinitiation complex
  • All these general transcription factors and pol II are enough to generate basal transcription
  • Transcription can be upregulated or downregulated by a huge diversity of other cis and trans acting factors to be discussed in chapter 12.
  • Once an mRNA is produced, it must be processed.
  • Processing involves the addition of a cap, the addition of a poly-A tail, and splicing out of introns.
rna processing mrna
RNA processing – mRNA
  • Transcription generates messenger RNA
    • A continuous sequence of nucleotides encoding a polypeptide
    • Transported to cytoplasm from the nucleus
    • Attached to ribosomes for translation
    • Are processed to remove noncoding segments
    • Are modified to protect from degradation and regulate polypeptide production
rna processing mrna23
RNA processing – mRNA
  • RNA polymerase II assembles a 1° transcript that is complementary to the DNA of the entire transcription unit
  • 1° transcript contains both coding (specify amino acids) and noncoding sequences
  • Subject to rapid degradation in its raw state
rna processing mrna24
RNA processing – mRNA
  • 5’ cap
  • 5' methylguanosine cap forms very soon after RNA synthesis begins
    • 1. The last of the three phosphates is removed by an enzyme
    • 2. GMP is added in inverted orientation so guanosine 5' end faces 5' end of RNA chain
    • 3. Guanosine is methylated at position 7 on guanine base while nucleotide on triphosphate bridge internal side is methylated at ribose 2' position (methylguanosine cap)
rna processing mrna25
RNA processing – mRNA
  • 5’ cap
  • Possible/known functions of 5’ cap
    • May prevent exonuclease digestion of mRNA 5' end,
    • Aids in transport of mRNA out of nucleus
    • Important role in initiation of mRNA translation
rna processing mrna26
RNA processing – mRNA
  • Polyadenlyation
  • The poly(A) tail – 3' end of most mRNAs contain a string of adenosine residues (100-250) that forms a tail
    • Protects the mRNA from degradation
    • AAUAAA signal ~20 nt upstream from poly(A) addition site
    • Poly(A) polymerase, poly(A) binding proteins, and several cleavage factors are involved
    • http://www.as.wvu.edu/~dray/219files/mRNAProcessingAdvanced.wmv
rna processing mrna27
RNA processing – mRNA
  • mRNA processing – Splicing
  • Requires break at 5' & 3' intron ends (splice sites) & covalent joining of adjacent exons (ligation)
      • http://www.as.wvu.edu/~dray/219files/mRNASplicingAdvanced.wmv
  • Why introns?
    • Disadvantages – extra DNA, extra energy needed for processing, extra energy needed for replication
    • Advantages – modular design allows for greater variation and relatively easy introduction of that variation
rna processing mrna28
RNA processing – mRNA
  • mRNA processing – Splicing
  • Splicing MUST be absolutely precise
  • Most common conserved sequence at eukaryotic exon-intron borders in mammalian pre-mRNA is G/GU at 5' intron end (5' splice site) & AG/G at 3' end (3' splice site)
rna processing mrna29
RNA processing – mRNA
  • mRNA processing – Splicing
  • Sequences adjacent to introns contain preferred nucleotides that play an important role in splice site recognition
review from last time30
Review from last time
  • Transcription cannot proceed until the pre-initiation complex has been constructed at the promoter
  • Construction of the pre-initiation complex is a stepwise recruitment process that eventually brings in RNA pol II
  • Multiple transcription factors are involved, know them and their functions
  • The primary transcript is capped almost immediately by a methylguanosine nucleotide that serves multiple functions
  • The 3’ end of the transcript is cleaved and a poly-A tail is added
  • Splicing of the primary transcript must be precise and multiple sequence-based landmarks aid the process
rna processing mrna31
RNA processing – mRNA
  • mRNA processing – Splicing
  • Nuclear pre-mRNA (common)
    • snRNAs + associated proteins = snRNPs
      • snRNAs – 100-300 bp
      • U1, U2, U4, U5, U6
    • 3 functions for snRNPs
      • Recognize sites (splice site and branch point site)
      • Bring these sites together
      • Catalyze cleavage reactions
    • Splicosome – the set of 5 snRNPs and other associated proteins
    • Summary movie available at:
    • http://www.as.wvu.edu/~dray/219files/mRNAsplicing.swf
rna processing mrna32
RNA processing – mRNA
  • mRNA processing – Splicing
  • 1. U1 and U2 snRNPs bind via complementary RNA sequences
  • Note the A bulge produced by U2
  • U2 is recruited by proteins associated with an exon splice enhancer (ESE) within the exon
rna processing mrna33
RNA processing – mRNA
  • mRNA processing – Splicing
  • 2. U2 recruits U4/U5/U6 trimer
  • U6 replaces U1, U1 and U4 released
  • U5 binds to upstream exon
rna processing mrna34
RNA processing – mRNA
  • mRNA processing – Splicing
  • 3. U6 catalyzes two important reactions
    • Cleavage of upstream exon from intron (bound to U5)
    • Lariat formation with A bulge on intron
  • Exons are ligated
  • U2/U5/U6 remain with intron
rna processing mrna35
RNA processing – mRNA
  • mRNA processing – Splicing
  • Several lines of evidence suggest that it is the RNA in the snRNP that actually catalyzes the splicing reactions
    • 1. Pre-mRNAs are spliced by the same pair of chemical reactions that occur as group II (self-splicing) introns
    • 2. The snRNAs needed for splicing pre-mRNAs closely resemble parts of the group II introns
  • Proteins likely serve supplemental functions
    • 1. Maintaining the proper 3D structure of the snRNA
    • 2. Driving changes in snRNA conformation
    • 3. Transporting spliced mRNAs to the nuclear envelope
    • 4. Selecting the splice sites to be used during the processing of a particular pre-mRNA
rna processing mrna36
RNA processing – mRNA
  • mRNA processing – Splicing
  • Group II intron self-splicing summary (rare)
rna processing mrna37
RNA processing – mRNA
  • Implications of RNA catalysis and splicing
  • The RNA world
    • Which came first, DNA or protein?... Apparently, it could have been RNA
    • Information coding AND catalyzing ability
  • Alternative splicing
        • Allows one gene to encode multiple protein products
    • Intron sequences actually encode some snoRNAs
  • Evolutionary innovation
    • Exon shuffling
rna processing rrna
RNA processing - rRNA
  • Eukaryotic ribosomes have four distinct rRNAs:
    • Three rRNAs in the large subunit (28S, 5.8S, 5S in humans);
    • One in the small (18S in humans) subunit
    • S value (or Svedberg unit)
      • 28S = ~5000 nucleotides
      • 18S = ~2000 nucleotides
      • 5.8S = ~160 nucleotides
      • 5S = ~120 nucleotides
    • RNA pol I transcribes all but 5S
    • 5S is transcribed by RNA pol III
ribosomal rna rrna
Ribosomal RNA - rRNA
  • Ribosomes are the location of protein synthesis
    • They are combinations of protein and RNA and are made up of two parts (small and large subunits)
  • Millions exist in any given eukaryotic cell
  • ~80% of RNA in a cell is rRNA
  • rDNA, typically exists in hundreds of tandemly repeated copies
rna processing rrna41
RNA processing - rRNA
  • The likely rRNA processing pathway
    • Cleavages 1 and 5 remove the ends of the 1° transcript
    • Two pathways exist for the remaining processing
    • End result is the same –
      • 18S + paired 28S/5.8S
    • 5S is produced by a second transcription unit
review from last time42
Review from last time
  • The splicosome is a complex of RNA and protein units responsible for splicing of immature mRNAs
  • Be able to describe the functions of each snRNP
  • The RNA portion of the snRNPs binds to the mRNA and to other snRNPs and actually catalyzes the splicing
  • The protein in the snRNPs serves other structural and functional roles
  • Ribosomal RNA is transcribed as a long unit but later chopped up to its constituent parts
  • The constituents along with proteins make up the small and large ribosomal subunits
rna processing 5s rna
RNA processing – 5S RNA
  • 5S rRNA
  • Transcribed by RNA pol III
  • Pol III is unique in that it utilizes promoters within the transcription unit
rna processing trna
RNA processing - tRNA
  • Transfer RNAs (tRNA)
  • Responsible for carrying amino acids to the site of protein synthesis
  • In humans, ~1300 genes for ~50 tRNAs
      • Human tRNA genes exist on all chromosomes except 22 and Y and are highly clustered on 1, 6, and 7
  • Transcribed by RNA pol III
rna processing
RNA processing
  • Small noncoding RNAs and RNA silencing
  • To study the effect of disabling a gene, researchers have had to produce ‘knockouts’ through a difficult, time consuming process involving some random chance.
  • …until the discovery of RNA interference
    • introduce dsRNA for the gene to be silenced and the mRNAs for that gene are destroyed
10 38 es cells jpg
10_38_ES.cells.jpg
  • …until the discovery of RNA interference
    • introduce dsRNA for the gene to be silenced and the mRNAs for that gene are destroyed
rna processing47
RNA processing
  • Mechanisms of RNA interference (siRNAs)
  • Dicer – RNA endonuclease
  • One of the RNA strands is destroyed, the other acts to identify the target mRNA as part of RISC complex
  • Slicer – RNA endonuclease portion of RISC
  • Likely a defense against foreign DNA
rna processing48
RNA processing
  • MicroRNAs (miRNA)
  • Work via a similar mechanism
  • Different source
  • Synthesized by RNA pol II
  • Later cleaved by dicer
  • Block translation
translation
Translation
  • By the end of this series of slides, you should be able to explain much of this animation
  • http://www.as.wvu.edu/~dray/219files/Translation_588x392.swf
  • An alternate animation is also provided: http://www.as.wvu.edu/~dray/219files/TranslationAdvanced.wmv
translation50
Translation
  • The genetic code
  • Amino acids in a protein are determined by a degenerate, triplet code
  • The code was determined using synthetic RNAs
  • The first, poly(U) -> polyphenylalanine
  • The genetic code is nearly universal
translation51
Translation
  • The genetic code
  • Codon assignments are nonrandom;
  • Codons for same amino acid tend to be similar
  • Benefits:
    • Less likely for a mutation to alter the amino acid sequence
      • Synonymous vs nonsynonymous mutations
    • Amino acids with similar chemical properties are encoded by similar codons
translation52
Translation
  • Translation - converting the nucleic acid information to amino acid information
  • A. Each tRNA is linked to a specific amino acid
  • B. Each tRNA is also able to recognize a particular codon in the mRNA
  • C. Interaction between successive codons in mRNA & specific aa-tRNAs leads to synthesis of polypeptide with an ordered amino acid sequence
translation53
Translation
  • tRNA characteristics
  • 1. All are relatively small with similar numbers of nucleotides (73 – 93)
  • 2. All have a significant number of unusual bases made by altering normal base posttranscriptionally
  • 3. All have base sequences in one part of molecule that are complementary to those in other parts
  • 4. Thus, all fold in a similar way to form cloverleaf-like structure (in 2 dimensions)
  • 5. Amino acid carried by the tRNA is always attached to A (adenosine) at 3' end of molecule
  • 6. Unusual bases concentrated in loops where they disrupt H bond formation; also serve as potential recognition sites for various proteins
translation55
Translation
  • Codon – Anticodon pairing
  • Similar to typical basepairing but allows for third position wobble
  • The first two positions must pair exactly but the third is more relaxed
  • Anticodon U can pair with A or G on mRNA
  • Anticodon I (derived from G) can pair with U, C, or A
  • Allows for fewer required tRNAs
    • Leucine (6 codons) requires only 3 different tRNAs
translation56
Translation
  • tRNA activation
  • Aminoacyl-tRNA synthetase (aaRS) guides charging of tRNAs with amino acids
  • ~20 different versions for the 20 different aa’s
translation57
Translation
  • Ribosome structure
  • Each ribosome has 3 sites for association with tRNAs; the sites receive each tRNA in successive steps of elongation cycle
    • A (aminoacyl) site -
    • P (peptidyl) site
    • E (exit) site -
  • A channel for the nascent polypeptide to exit is also present
translation58
Translation
  • Ribosome structure
  • tRNAs bind within these sites & span the gap between the 2 ribosomal subunits
    • The anticodon ends of the bound tRNAs contact the small subunit, which plays a key role in decoding the information contained in the mRNA
    • The amino acid-carrying ends of bound tRNAs contact the large subunit, which plays a key role in catalyzing peptide bond formation
review from last time59
Review from last time
  • 5S rRNA and tRNAs are transcribed by RNA pol III
  • RNA pol III is unique in its use of an internal promoter
  • siRNAs and miRNAs are RNAs involved in shutting down a gene’s function without affecting the gene itself
  • The genetic code is degenerate
  • tRNAs are short RNAs that bridge the gap between information in the mRNA and amino acid chain
  • The structure of a ribosome is such that three sites are formed, A, P, and E
  • As mRNA threads through the ribosome the information encoded is translated to form an amino acid chain
translation60
Translation
  • Initiation of translation
  • Step 1. Bind the initiation codon (AUG, met) to the small ribosomal subunit
  • In bacteria
  • The Shine-Dalgarno sequence on mRNA is complementary to 16S rRNA
  • Initiation Factors
    • IF1 – attaches 30S subunit to mRNA
    • IF2 – required for attachment of first tRNA
    • IF3 – likely prevents bind of large subunit
translation61
Translation
  • Initiation of translation
  • Step 2. Bind the first tRNA (tRNAMet)
  • Enters the P site with the help of IF 2
translation62
Translation
  • Initiation of translation
  • Step 3. Bind the large subunit
  • IFs released and large subunit binds
translation63
Translation
  • Initiation of translation
  • Bind the initiation codon (AUG, met) to the small ribosomal subunit
  • In eukaryotes
  • Three IFs + tRNAMet bind to 40S subunit
  • Separately mRNA binds to additiona IFs and PABP
  • These components come together and scan along mRNA until AUG is reached
  • Large subunit binds
translation64
Translation
  • Elongation
  • The players – EF-Tu/GTP/tRNA complex
    • EF-Tu – escorts the tRNA to the A site
    • GTP – provides energy
    • The tRNA - duh
  • Any tRNA can enter but only the correct one will induce the conformational changes to induce binding to mRNA
  • Once in, GTP -> GDP and Tu-GDP is released
translation65
Translation
  • Elongation
  • Peptide bond is formed between aa’s
  • Peptidyl transferase – a ribozyme
  • tRNA in P site is now uncharged
translation66
Translation
  • Elongation
  • Translocation of the ribosome along the mRNA (3 nt)
  • tRNAs rachet positions
  • EF-G induced
  • GTP -> GDP + P required
translation67
Translation
  • Elongation
  • Release of the uncharged tRNA and repeat the whole cycle
  • ~1/20 second
translation68
Translation
  • Termination
  • Three codons (UAA, UGA, UAG) have no complementary tRNAs
  • Protein released when one is reached
  • Release factors are required
  • Bacteria RF1, RF2, RF3
  • Eukaryotes eRF1, eRF3
  • Each recognizes particular stop codon much like a tRNA
  • RF3/eRF3 binds GTP to energize the release of the polypeptide and disassemble the ribosome
  • The complete process (for bacteria) is illustrated using videos on the class website.
translation69
Translation
  • Polyribosomes

Prokaryote

Eukaryote

Note the difference –

Due to presence/absence of

nuclear membrane

slide70

http://www.as.wvu.edu/~dray/219files/Protein_Synthesis%20_Translation_2008.avihttp://www.as.wvu.edu/~dray/219files/Protein_Synthesis%20_Translation_2008.avi