Review from last time

1
Review from last time

• Gene duplication occurs much more often than
  genome duplication
• Gene duplication can provide a source of
  variation for the development of new functions in
  organisms
• Transposable elements are interspersed sequences
  in all eukaryotic genomes
• Be familiar with the structure and mobilization
  mechanisms for class 1 and class 2 elements
• Be able to describe the potential impacts of
  mobile elements on a genome
• The most current estimate is 25-30,000 genes in
  our genome
• Comparative genomics can provide information on
  the similarities and differences among genome and
  indicate what parts are important

2
Chapter 11Gene Expression From Transcription
to Translation
3
This Chapter in One Slide
4
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

5
(No Transcript)
6
Transcription

• Transcription the process of copying a DNA
  template into an RNA strand
• Accomplished via DNA dependent RNA polymerase
  (aka RNA polymerase)

7
Transcription

• 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/Transcription
  _588x392.swf

8
Transcription

• Begins with the association of the RNA polymerase
  with the DNA template
• Which brings up DNA protein interactions
• Some enzymes have evolved to recognize specific
  DNA sequences
• One such DNA sequence is called a promoter
• The promoter is the assembly point for the
  transcription complex
• RNA polymerases cannot recognize promoters on
  their own, but require the help of other proteins
  (transcription factors)

9

• Bacterial RNA polymerase can incorporate 50 - 100
  nucleotides/sec
• Most genes in cell are transcribed simultaneously
  by numerous polymerases
• Polymerase moves along DNA in 3' gt 5' direction
• Complementary RNA constructed in 5' gt 3'
  direction
• RNAn NPPP gt RNAn1 PPi

10
Transcription

• Prokaryotic Transcription
• One type of RNA polymerase with 5 subunits
  tightly associated to form core enzyme
• Core enzyme minus sigma (s) factor will bind to
  any DNA.
• By adding s, RNA pol will bind specifically to
  promoters

11
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

12
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
• s factors and polymerases recognize the sequences
  and bind to them

TTGATA TTGACA CTGACG
13
Transcription

• Eukaryotic Transcription
• 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)

14
Transcription

• Eukaryotic 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)

15
Transcription

• Eukaryotic 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)

16
Transcription

• Eukaryotic Transcription
• All major RNA types (mRNA, tRNA, rRNA) must be
  processed
• The final products are derived from precursor RNA
  molecules that are considerably longer than the
  final RNA product
• The primary (1) transcript is is equivalent in
  length to the full length of the DNA transcribed
• The corresponding segment of DNA from which 1
  transcript is transcribed is called transcription
  unit
• The1 transcript is short-lived it is processed
  into smaller, functional RNAs
• Processing requires variety of small RNAs (90
  300 nucleotides long) their associated proteins

17
Review from last time

• Chapter 11 is about two processes
• Transcription the process of copying a DNA
  strand into RNA
• Translation the process of producing an amino
  acid chain from a transcribed RNA
• RNA is similar to DNA but with some minor
  differences
• There are several different types of RNA
• Without RNA, there can be no gene expression
• The promoter is the site of assembly of the
  transcription apparatus, be familiar with it
• Promoters are particular DNA sequences that are
  bound by transcription factors
• Prokaryotic RNA polymerase complexes consist of
  five components sigma specifies the promoter
  sequence used
• Eukaryotic transcription is more complex
• More components
• Three different RNA polymerases with different
  jobs
• In eukaryotes, RNA transcripts must be processed

18
RNA processing

• 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

19
RNA processing
20
RNA processing

• 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

21
RNA processing

• Eukaryotic ribosomes have four distinct rRNAs
• 28S, 5.8S 18S rRNAs are produced from a single
  1 transcript that is transcribed by RNA pol I
• 5S rRNA is synthesized from a separate RNA
  precursor using RNA pol III

22
RNA processing

• 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

23
RNA processing

• snoRNAs small nucleolar RNA
• Vital to rRNA processing
• Pair with proteins to make snoRNPs
• Consist of relatively long stretches (10-21
  nucleotides) that are complementary to parts of
  rRNA transcript
• can form double-stranded hybrids
• bind to specific portions of pre-rRNA to form an
  RNA-RNA duplex guide an enzyme within the
  snoRNP to modify a particular pre-rRNA nucleotide
• 200 different snoRNAs exist

24
RNA processing

• snoRNAs small nucleolar RNA
• snoRNPs associate with rRNA precursor before it
  is fully transcribed
• Best characterized RNP contains U3 snoRNA and gt2
  dozen different proteins
• Binds to precursor 5' end of transcript
  catalyzes removal of transcript 5' end

25
RNA processing

• 5S rRNA
• Transcribed by RNA pol III
• Pol III is unique in that utilizes promoters
  within the transcription unit

26
RNA processing

• 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

27
RNA processing

• 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

28
RNA processing

• 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

29
RNA processing

• 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)

30
RNA processing

• 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

31
RNA processing

• 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.

32
Review from last time

• All RNA transcripts must be processed.
• 3 of the 4 ribosomal RNAs (rRNAs) are transcribed
  as a single unit and processed by cleaving
  individual units out
• snoRNAs are critical to the rRNA processing
• tRNAs and 5S rRNA are transcribed by RNA pol III
• RNA pol III genes are unique in having internal
  promoters
• Be aware of the components making up the
  preinitiation complex of a RNA pol II gene and
  their roles in transcription initiation
• Review of RNA pol II transcription initiation at
• http//www.as.wvu.edu/dray/219files/Transcription
  Advanced.wmv
• Review of human genome complexity at
• http//www.dnalc.org/ddnalc/resources/chr11a.html

33
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

34
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

35
RNA processing

• mRNA processing 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)

36
RNA processing

• mRNA processing 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

37
RNA processing

• mRNA processing 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/mRNAProcessin
  gAdvanced.wmv

38
RNA processing

• 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/mRNASplicingA
  dvanced.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

39
RNA processing

• 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)

40
RNA processing

• mRNA processing Splicing
• Sequences adjacent to introns contain preferred
  nucleotides that play an important role in splice
  site recognition

41
RNA processing

• 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

42
Review from last time

• Messenger RNAs (mRNAs) experience three
  processing steps
• Addition of a methylguanosine cap
• Polyadenylation
• Splicing
• Be familiar with the characteristics and
  functions of the 5 cap
• Be able to describe the polyadenylation signals
  on an mRNA, the functions of the proteins
  involved, and the process of polyadenylation
• Be able to describe the nature of the splicosome
• Be able to describe the sequence landmarks
  required for accurate splicing

43
RNA processing

• 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

44
RNA processing

• mRNA processing Splicing
• 2. U2 recruits U4/U5/U6 trimer
• U6 replaces U1, U1 and U4 released
• U5 binds to upstream exon

45
RNA processing

• 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

46
RNA processing

• 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

47
RNA processing

• mRNA processing Splicing
• Group II intron self-splicing summary (rare)

48
RNA processing

• 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

49
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

50
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
51
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

52
RNA processing

• MicroRNAs (miRNA)
• Work via a similar mechanism
• Different source
• Synthesized by RNA pol II
• Later cleaved by dicer
• Block translation

53
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_5
  88x392.swf
• An alternate animation is also provided
  http//www.as.wvu.edu/dray/219files/TranslationAd
  vanced.wmv

54
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) -gt polyphenylalanine
• The genetic code is nearly universal

55
Review from last time

• The splicosome is a complex of multiple snRNPs
• Be familiar with the model of splicosome function
  in removing introns
• Arguments for RNA-based early life were bolstered
  by the discovery that RNA can catalyze reactions
  independently of protein
• The difficult process of discovering gene
  function by producing knockouts can be
  circumvented using RNA interference
• Be able to describe the differences in the
  function of microRNAs and siRNAs
• The genetic code is a triplet code, you should be
  able to describe why that is and determine what
  amino acids are encoded by a given RNA strand

56
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

57
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

58
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

59
(No Transcript)
60
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

61
Translation

• tRNA activation
• Aminoacyl-tRNA synthetase (aaRS) guides charging
  of tRNAs with amino acids
• 20 different versions for the 20 different aas

62
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 16 rRNA
• Initiation Factors
• IF1 attaches 30S subunit to mRNA
• IF2 required for attachment of first tRNA
• IF3 likely prevents bind of large subunit

63
Translation

• Initiation of translation
• Step 2. Bind the first tRNA (tRNAMet)
• Enters the P site with the help of IF 2

64
Translation

• Initiation of translation
• Step 3. Bind the large subunit
• IFs released and large subunit binds

65
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

66
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

67
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

68
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 -gt GDP and Tu-GDP is released

69
Translation

• Elongation
• Peptide bond is formed between aas
• Peptidyl transferase a ribozyme
• tRNA in P site is now uncharged

70
Translation

• Elongation
• Translocation of the ribosome along the mRNA (3
  nt)
• tRNAs rachet positions
• EF-G induced
• GTP -gt GDP P required

71
Translation

• Elongation
• Release of the uncharged tRNA and repeat the
  whole cycle
• 1/20 second

72
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.

73
Translation

• Polyribosomes

Prokaryote
Eukaryote
Note the difference Due to presence/absence
of nuclear membrane