7' Expression of Genetic Information 2

1
7. Expression of Genetic Information (2)
2
Relationship between Genes and Proteins

• Transcription tRNA Processing
• 50 different tRNAs
• Each coded by DNA
• Human 1300 repeats
• Genes in small clusters
• tDNA
• Large non-transcribed spacer sequences
• tRNA coding sequences irregularly spaced
• In tandem repeats

3
Relationship between Genes and Proteins

• Transcription tRNA Processing
• Transcribed by RNA polymerase III
• Promotor sequence within the gene
• Primary transcript larger than final product
• 5 and 3 ends and some central sequences trimmed
• All mature tRNAs have the triplet CCA at 3 end
• Added enzymatically after tRNA is processed
• Key role in protein synthesis

4
Relationship between Genes and Proteins

• Transcription mRNA Processing
• Mechanism
• All mRNA precursors synthesized by RNA polymerase
  II
• An enzyme composed of 12 subunits
• Highly conserved across species
• Initiation of transcription with cooperation of
  general transcription factors (GTFs)
• Same GTFs used for a wide range of mRNAs

5
Relationship between Genes and Proteins

• Transcription mRNA Processing
• Mechanism
• All mRNA precursors synthesized by RNA polymerase
  II
• Promoters to the 5 side of transcription units
• Critical portion of promoter core promoter
  element
• Lies between 24 32 bases upstream of
  transcription initiation site
• Consensus sequence 5-TATAAAA-3 (TATA box)
• resides on the coding strand of DNA
• not on the template strand

6
Relationship between Genes and Proteins

• Transcription mRNA Processing
• Mechanism
• TATA box
• Site of assembly of preinitiation complex
  containing GTFs and polymerase
• Assembled prior to transcription initiation

7
Relationship between Genes and Proteins

• Transcription mRNA Processing
• Mechanism
• TATA box
• Site of assembly of preinitiation complex
  containing GTFs and polymerase
• Assembled prior to transcription initiation
• TATA binding protein
• Recognizes TATA box
• Binding of TBP to polymerase causes distortion of
  DNA
• Inserts into double helix
• Bends DNA more than 80º at the interaction site

8
Relationship between Genes and Proteins

• Transcription mRNA Processing
• Mechanism
• RNA polymerase II large subunit C-terminal domain
• -Tyr-Ser-Pro-Thr-Ser-Pro-Ser-
• In mammals 52 repeats
• Phosphorylation by protein kinases
• Preinitiation complex not phosphorylated
• During transcription it is heavily phosphorylated
  all in C-terminal
• Trigger uncouples enzyme from GTFs/promoter
  move down DNA template

9
Relationship between Genes and Proteins

• Transcription mRNA Structure
• Common properties
• Continuous sequences of nucleotides coding
  polypeptides
• Found in the cytoplasm
• Attached to ribosome when translated
• Most contain a non-coding segment
• Have important regulatory roles
• Specially modified 5 3 termini
• 3 end with a string of adenosine residues
  (poly(A) tail)
• 5 end methylated guanosine cap

10
Relationship between Genes and Proteins

• Transcription mRNA Structure
• Split genes
• Most genes are split containing large
  untranscribed sequences
• Part of the gene contributing to mature mRNA is
  termed an exon
• Part of gene which is not transcribed is termed
  an intron
• Introns found in all genes rRNA, tRNA, mRNA

11
Relationship between Genes and Proteins

• Transcription mRNA processing
• Primary transcript complementary to the DNA of
  the entire transcription unit
• Transcript processed in the nucleus to mature
  mRNA
• Transported to cytoplasm
• mRNA transcripts associated with proteins and
  larger particles during translation
• Particles
• Proteins, ribonucleoproteins
• Convert primary transcript into mature mRNA

12
Relationship between Genes and Proteins

• Transcription mRNA processing
• Conversion
• Addition of 5 cap
• 3 poly(A) tail
• Removal of introns

13
Relationship between Genes and Proteins

• Transcription mRNA processing
• Addition of 5 caps
• 5 ends initially possess a triphosphate
• Once synthesized, several enzymes act
• Last phosphate removed gives a diphosphate

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Relationship between Genes and Proteins

• Transcription mRNA processing
• Addition of 5 caps
• A GMP added in inverted orientation
• Terminal inverted guanosine is methylated at the
  7 position
• Ribose on base 1 methylated at the 2 position
• 5 end of RNA now has a methylguanosine cap

15
Relationship between Genes and Proteins

• Transcription mRNA processing
• Addition of 5cap
• Functions
• Prevents 5 end from digestion by exonucleases
• Transport of RNA out of nucleus
• Role in initiation of translation

16
Relationship between Genes and Proteins

• Transcription mRNA processing
• Addition of 3poly(A) tail
• Begins approx 15 nucleotides downstream from
  sequence AAUAAA
• Recognition site for assembly of protein complex
• Processing reactions at 3 end
• Includes an endonuclease cleaves pre-mRNA
  downstream from recognition site
• Following cleavage an enzyme
  poly(A)-polymerase adds approx 250 adenosines
  without need of a template

17
Relationship between Genes and Proteins

• Transcription mRNA processing
• mRNA removal of introns from pre-mRNA
• Complex process known as RNA splicing
• Processing occurs as each intron becomes
  associated with a complex termed a splicosome
• snRNPs
• Range of proteins

18
Relationship between Genes and Proteins

• Transcription mRNA processing

19
Relationship between Genes and Proteins

• Transcription mRNA processing
• mRNA removal of introns from pre-mRNA