Identifying ORFs depends on searching for start and stop codons'

1

• Identifying ORFs depends on searching for start
  and stop codons.
• Confirmation that an ORF is discovered depends on
  the location of a promoter.
• Failure to find a promoter suggests that start
  and stop codons are part of an operon.

2

• In prokaryotes, the ribosome binds to nts 5 to
  the start site of translation.
• These nts are called Shine-Delgarno sequences.
• Typically the sequence is 5 AGGAGGU 3
• Mutations in the Shine-Delgarno sequence can
  prevent mRNA from being translated.
• The Shine-Delgarno sequence is an important
  marker for a ORF

3

• A common practice is to use computer software to
  generate a translation from a DNA or mRNA
  sequence.
• The process is called conceptual translation.
• The program reads the DNA or mRNA in terms of
  codons.
• Generates the protein sequence

4

• The protein sequence can be compared to other
  known protein sequences in a proteomics database
• Operons contain specific sequences for the
  termination of transcription.
• These sequences are called intrinsic terminators

5

• Intrinsic terminators have two features
• (1) an inverted repeat, i.e.
• 5-CGGATGCATCCG-3
• 3-GCCTAC GTAGGC-5
• (2) approximately 6 uracils following the
  inverted repeat

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• RNA molecules can form secondary structure.
  Usually due to base pairing within the inverted
  repeats.
• Typically intrinsic terminators the inverted
  repeat is 7 20 nts in length and rich in GCs.
• The significance of the secondary structure is
  that it causes the RNA polymerase to pause about
  1 minute
• This is a very long time since the rate of
  incorporation is 100 nts/sec

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• If the sequence was between uracil and adenine
  (DNA template) Then the nascent mRNA would
  dissociate from the template and effectively end
  transcription.
• So the pause is sufficient to terminate
  transcription.

8

• The relative abundance of GC versus AT in a
  bacterial genome is a distinguishing attribute of
  bacterial genomes
• Measuring GC content can be used to identify
  bacteria.
• GC content varies from 25 to 75 across
  different prokaryote genomes.
• The GC content of bacteria is altered over time
  by DNA polymerase and repair mechanisms

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• This leads to ratios of GC and AT regions within
  a bacteria genome being uniform.
• Studying GC content has revealed that bacteria
  genes have been transferred as groups not single
  genes.
• These sequences range in length from hundreds to
  thousands of nts.
• The process of transferring genes is known as
  horizontal gene transfer.
• This leads to bacterial genomes appearing to be
  patchworks of regions with distinctive GC
  content.

10

• In the bacterial genome between 85 - 88 of the
  nts are associated with coding genes.
• In E. coli, the average coding sequence length is
  950 bp and these sequences are separated by only
  118bp.
• It is theorized that having such a compact genome
  may aid in the ability to quickly divide.
• It is interesting that not only can bacteria
  acquire large GC sequences but they can loose
  them as well.

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• Bacterial genomes have the following
  characteristics
• Long ORF (60 or more codons in length)
• Matches to simple promoter sequences, RNA
  polymerase recognize the -35 and -10 sequences of
  promoters.
• Recognizable transcriptional termination signal

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• Comparisons with nts (amino acids) with other
  bacterial species is very good
• Eukaryotic genomes are very different than
  prokaryotic genomes.
• Compartmentalization of cellular functions means
  more control is required.
• Most eukaryotes are multicellular and though they
  have the same genome. The expression of genes is
  different.

13

• Eukaryote genomes have more nts than seems to be
  required for genes and control sequences, i.e.
  junk DNA.
• This leads eukaryote genomes to be more complex
  and flexible than prokaryote genomes.
• Eukaryote genomes have two copies of each gene.
  In humans, the autosomes chromosomes 1 22 and
  the sex chromosomes
• The size of the chromosomes is greater than
  prokaryotes.

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• The shortest chromosome is 55Mbs and the longest
  chromosome is 3,200 Mbs.
• Sequencing eukaryote genomes is more difficult
  than prokaryote genomes.
• Simply searching for overlapping fragments is not
  sufficient for sequencing eukaryote genomes.
• The use of STS (sequence tagged sites) and EST
  (expressed sequence tags)
• These sequences are determined from a physical
  map of the chromosome and then used to arrange
  the DNA fragments

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• Discovering eukaryote genes can be accomplished
  using software such as
• Grail EXP and GenScan which rely on neural
  networks and dynamic programming techniques.
• Neural networking relies on searching for
  statistical similarities between a known sequence
  and unknown sequence.
• These programs search for intron/exon boundaries,
  promoters, and putative ORFs

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• Prokaryotes use a single RNA polymerase
• While eukaryotes have three different RNA
  polymerases which are comprised of 8 to 12
  proteins.
• Each eukaryote RNA polymerase recognizes a
  different promoter.
• RNA polymerase I has promoter location -45 to 20
  and transcribes genes for ribosomal RNAs

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• RNA polymerase II binds to promoter far upstream
  to -25. The transcribed genes are
  protein-coding.
• RNA polymerase III binds to promoter 50 to 100.
  The transcribed genes are tRNA and other small
  RNAs.
• In eukaryotes, every gene has its own promoter.
  Unlike prokaryotes which can have one promoter
  for multiple genes.

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• RNA polymerase II promoters contain a sequence
  known as the basal promoter.
• This is the location where the RNA polymerase II
  initiation complex is assembled and transcription
  begins.
• In addition, there are upstream promoter elements
  to which proteins other than RNA polymerase II
  bind.
• It is estimated that 5 upstream promoter elements
  are required to uniquely identify any particular
  gene.
• Note, assembly of the initiation complex can
  occur with only the core promoter without the
  proteins associated with the upstream elements
  but it is inefficient.

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• RNA polymerase II does not bind directly to the
  basal promoter unlike the prokaryote RNA
  polymerase.
• Instead basal transcription factors bind to the
  RNA polymerase II and the promoter.
• These proteins include a TATA-binding protein
  (TBP) and at least 12 TBP-associated factors
  (TAF)
• Just like prokaryotes, eukaryotes have a TATA box
• The sequence is 5-TATAWAW-3 where W is a either
  a A or T at same frequency.

21

• The transcription start site (1) is associated
  with an initiator (Inr) sequence
• 5-YYCARR-3 where Y C or T and R G or A
• The 1 nt is almost always A.
• Differences in basal transcription factors exist
  between cell types in the same organism.
• This leads to differential expression of genes
  according to cell type.

22

• The presence of Inr and TATA box are important
  eukaryotic markers for putative genes.
• In prokaryotes, the RNA polymerase has a strong
  affinity for the promoter. The same is not true
  for eukaryotes.
• Eukaryotes have a low rate of transcription
  irregardless of the promoter sequence.
• So eukaryotes depend on proteins that act as
  positive regulators to help in transcription.

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• Some of these positive regulatory proteins are
  constitutive. That is they work on various genes
  and do not respond to external signals.
• While others are regulatory. These interact with
  specific genes and do respond to external
  signals.
• Collectively these proteins are called
  transcription factors.
• Most transcription factors bind to specific DNA
  sequences

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• Some transcription factors recognize consensus
  sequences close to the transcription start.
• A good example is the CAAT box at position -80 in
  most eukaryotic gene. This sequence is always
  found in the same orientation.
• While others are located further away from the
  transcription start site. These are generally
  called enhancers.
• Enhancers are not orientation specific and can be
  -500 to 500 from the transcription start site.

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• Interestingly, some enhancers are located tens of
  thousands of nts upstream from the transcription
  start site.
• These enhancers bend the DNA into specific shape
  that brings the transcription factors together.
• This is called an enhanceosome.
• Other enhancers help mediate response to heat,
  control differential gene expression in different
  cell types or during development.

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• The nucleus is physical barrier that allows the
  RNA transcript to be modified before it reaches
  the ribosome.
• These unmodified RNA transcripts are called hnRNA
• The modifications are capping, slicing and
  polyadenylation
• Capping occurs at the 5 end (includes
  methylation) and is performed on all hnRNA.
• Splicing is the removal of introns and connecting
  of exons
• Polyadenylation is the placement of 250 Adenines
  at the 3 end of hnRNA
• These modifications are cell type specific.

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• A major difficulty for the Bioinformatician is
  the ORF length does not suggest a protein coding
  sequence.
• Splicing can remove stop codons in the introns.
• A possibility is to search for exon/intron
  boundaries. However, the rules for splicing can
  vary according to cell type.

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Introns and Exons

• 8 different kinds of introns have been found in
  eukaryotes
• The GU-AG rule is useful for predicting protein
  coding sequences.
• In the GU-AG rule, the first two nts at the 5
  end of an intron are GU and AG is at the 3 end.
• There is also an internal branch site located
  18 to 40 bp upstream of the 3 splice junction.
• Most information introns is within the intron
  itself and not the exon

30

• Typically an intron has a minimal length of 60
  nts. There does not seem to be a maximum length.
• Exons can vary in length from 450 nts to over
  2000 nts. There some examples of exons less than
  100 but these are not the rule.
• Genes do not have a minimum number of introns.
  Some genes have over 100.
• Thus the length and number of introns does not
  seem important.
• However the position of the introns in genes is
  highly conserved. Suggesting that it is important

31

• The 5 and 3 splice junctions seem to be the
  same. Yet is known that in some genes splicing
  occurs within only one intron.
• It is not known how the splicing proteins can
  differentiate between different 5 and 3 splice
  junctions.
• It doe not seem to based on the hnRNA sequence.

32

• Most hnRNA is spliced into one type of mRNA for
  all of the genes coding for the mRNA.
• Estimates are that 20 of the genes are
  alternatively spliced.
• The actual molecules involved in splicing are
  snRNA (small nuclear RNA) and additional
  proteins.
• It is possible that the variability in consensus
  sequences of splice junctions and branch sites
  maybe necessary for different snRNAs

33

• GC content in eukaryotic genomes is not as varied
  in prokaryotes.
• Computer algorithms use GC content for the
  following reasons
• There is correlation between GC content and
  genes, upstream promoter sequences, codon choice,
  gene length and gene density

34

• In eukaryote genomes the sequence CG occurs only
  20 of the expected frequency based on chance
  alone.
• Genes were found that had a 1 -2 kb sequence of
  GCs at the 5 end.
• These sequences were located at -1500 500 in the
  gene.
• These GC sequences are called CpG islands

35

• Many of the GCs are part of the binding sites for
  transcription factors, such as Sp1.
• In the human genome, there are 45,000 CpG islands
  and half are associated with known house-keeping
  genes.
• House-keeping genes are expressed in all tissues
  and at high levels.
• While others are associated with promoter
  sequences

36

• CpG islands are associated with methylation
• In methylation, DNA methylase attaches methyl
  groups to cytosine.
• The cytosine must occur as 5-CG-3 dinucleotides
• Methylated cytosine have a high rate of mutations
• High levels of methylated CG is associated with
  low levels of acetylated histones and vice versa

37

• High levels of acetylated histones is associated
  with gene expression.
• Methylated cytosines can suppress transcription
  when located in promoter sequences
• Interestingly methylation patterns vary from one
  cell type to another.

38

• Histones are positively charged proteins that
  have a high affinity for DNA (negatively charged)
• The positive charge on histones is reduced by the
  addition of acetylation.
• Histones whose affinity for DNA is less allows
  the DNA to be more accessible to RNA polymerase.

39

• Areas of active transcription are euchromatin
• Areas of inactive transcription are
  heterochromatin
• Heterochromatin is densely packed DNA and
  euchromatin is the opposite