Ch. 17 Transcription, RNA Processing, and Translation

• Transcription overview

  • RNA polymerases synthesize an mRNA version of the info stored in DNA
  • Uses ribonucleoside triphosphates (NTPs), do not need a primer to start
  • matches complementary bases to one strand of DNA, except with U instead of T
  • RNA is synthesized the the 5' to 3' direction
  • Only one strand of DNA is called the template strand
• • The non template (coding) strand is the strand that is not being used to synthesis the new strand
  • The template strand is where the RNA connects
  • Hydrogen bonds from between complementary base pairs
  • NTPs- nucleoside triphosphates monomers are added
  • Phosphodiester linkage is formed by RNA polymerase after base paring occurs
• Transcription

  three stages

  • initiation
  • elongation
  • termination
• Transcription in bacteria

  RNA polymerase can not start transcription on its own

  • in bacteria, sigma protein must bind to the core enzyme first
  • sigma can bind only in one orientation, so orientation of binding determines which DNA strand will be used as a template
  • RNA polymerase and sigma protein form a holoenzyme
• Transcription in Bacteria

  Sigma binds to DNA segments called promoters that promote the starts of transcription (tells RNA polymerase where to start)

  • different sigma proteins bind to promoters with slightly different DNA base sequences
  • The TATAAT box is upstream of the sigma protein
  • Downstream DNA is after the sigma proteins and is the way that DNA polym is synthesized
• Bacterial Promoters

  • are 40-50 base pairs long
  • have a specific sequence upstream of the transcription start site that sigma factor recognizes
  • have a -10 box TATAAT and a - 35 box TTGACA
  • The promoter is never transcribed
• 1. Initiation begins- sigma binds to promoter region of DNA
  2. RNA polymerase opens (no primase or helicase) the DNA helix; transcription begins by adding incoming NTPs to the new strand
  3. Elongation is starting- sigma is released from the promoter, RNA synthesis continues
  4. RNA polymerase transcribes a transcription-termination signal, which codes for RNA that forms a hairpin (complementary base)
  5. Termination- the RNA hairpin causes the RNA to separate from the RNA polymerase, terminating transcription
• Transcription in Eukaryotes

  • several differences
  • different DNA polymerases (mRNA is produced by RNA polymerase II)
  • more diverse promoters, including TATA box (about 30 base pairs upstream from start)
  • Basal Transcription Factors instead sigma proteins
  • At termination poly (A) signal- like 200 As are transcribed rather than a hairpin and the RNA transcript is released 10-35 nucleotides past this sequence
  • Transcription occurs in the nucleus
• Transcription factors in Eukaryotes

  1. Eukaryotic promoter
  2. Several transcription factors bind to DNA- a bundle of proteins
  3. Transcription initiation complex forms- there is no proofreading but it's fine because it only codes for proteins
• RNA processing in Eukaryotes

  • in bacteria mRNAs are translated immediately in cytosol even before transcription is complete
  • In eukaryotes the initial product of transcription is an immature primary transcript or pre-mRNA
  • Primary transcripts must undergo RNA processing before they can be translated
  • 2 events: RNA splicing, adding caps and tails to transcripts
• RNA splicing

  Exons- coding regions of eukaryotic genes that will be part of the final mRNA product- stay

  Introns- intervening non-coding sequences that will not be in the final mRNA- spliced out

  • In a micrograph picture, RNA is shorter than DNA because parts of RNA are spliced out
• RNA splicing

  • Introns are removed by splicing, catalyzed by small nuclear ribonucleoproteins or snRNPs, which form a complex called a spliceosome
  • Splicing allows different mRNAs and proteins to be produced from a single gene
• snRNPs splice RNA within the nucleus

  1. snRNPs bind to start of intron and a base within the intron
  2. snRNPs assemble to form the spliceosome
  3. Intron is cut - not necessary for protein and a loop forms
  4. Intron is released as a lariat- lasso, and exons are joined together
• Adding Caps and Tails to transcripts

  • A 5' cap- a modified guanine nucleotide enables ribosomes to bind and protects from degradation
  • 3' poly(A) tail- 100-250 adenine nucleotides; is needed for translation and protects from degradation
  • mature mRNAs contain Untranslated regions (UTRs) at both ends
• Translation

  ribosomes and tRNA is used

  • In bacteria start before transcription is complete. Multiple ribosomes attached to an mRNA before transcription to form a polyribosome, many copies of a protein are produced from one mRNA
  • In eukaryotes transcription and translated are separated, polyribosomes form too
• Transfer RNA (tRNA)

  • transfers amino acids to ribosome and matches them with codon
  • 75-85 nucleotides long, from secondary structure by folding into a stem-and loop
  • A CCA sequence (SER) at the 3' end is the building site for amino acids
  • The loop at the opposite end forms the anticodon- where amino acid attaches. Base pair with the mRNA codon
  • tRNA twists and folds into 3D upside down L-shape
  • amino acids are read 5' to 3', so mRNA fits 3' to 5'
• Transfer RNA (tRNA)

  • ATP is required to attach tRNA to an amino acid
  • Enzymes called aminoacyl-tRNA synthetases "charge" the tRNA by catalyzing the addition of amino acids to tRNAs
  • for each of 20 amino acids there is a different aminoacyl-tRNA synthetase, and more than 1 tRNA
  • there are 61 different codons but only about 40 tRNAs
• Wobble Hypothesis


  flexible pairing at the third base allows some tRNAs to bind to more than 1 codon

  eg: identical leucine tRNAs with a GAG sequence can bind to CUC or UUC
• Ribosomes

  ribosome contain different proteins and ribosomal RNA (rRNA)

  • The large subunit is where peptide bonds form
  • The small subunit hold the mRNA in place
• Three sites in the ribosome (three tRNAs can be bound at the same time)- APE

  1. The A site, acceptor site- tRNA carrying the correct anticodon for the mRNA codon enters the A site, an aminoacyl tRNA
  2. The P site, peptidyl site where a peptide bond forms between the amino acid on the A-site tRNA and the polypeptide on P-site tRNA, hold the tRNA with the growing peptide attached
  3. The E site, is where tRNA without amino acids exit the ribosome is. Ribosome moves down the mRNA by one codon and tRNAs move down one position
• Translation

  Three stages are 1. Initiation 2. Elongation 3. Termination

  • Amino acids are always added to the carboxyl (C-terminus) 3' end. mRNA is read 5' to 3' and amino acids are made 5' to 3'. N-terminus amine group, to C-terminus carboxyl group
  • all three stages require protein "factors" that aid in the translation process
  • Energy, ATP is needed
• Initiating Translation

  1. Initiation factors cause mRNA to bind to small subunit of ribosome; ribosome binding site is ahead (near 5') of start codon
  2. Initiator aminoacyl tRNA binds to start codon, holding f- MET
  3. Large subunit of ribosome binds the tRNA with f- MET in the P site
• Initiation contin.

  • the initiation phase of translation begins near the AUG start codon
  • The small ribosomal subunit binds to the mRNA at the ribosomal binding site (Shine Dalgarno sequence) about 6 bases upstream from the start codon, and is meditated by initiation factors
  • the first tRNA is called the initiator tRNA, it carries a modified methionine (f- MET) in bacteria
• Translation Elongation

  At the start of elongation the initiator tRNA is in the P site and the E and A sites are empty

  1. An aminoacyl tRNA bind to the codon in the A site
  2. The active site on the ribosome is entirely ribosomal RNA. It catalyzes peptide bond formation, the ribosome is an enzyme called ribozyme
  3. the amino acid of the P-site tRNA is transferred to the aminoacid on the A site tRNAincoming aminoacyl tRNA,
• Translation- Elongation

  At the start of elongation the initiator tRNA is in the P site and the E and A sites are empty

  • An aminoacyl tRNA bind to the codon in the A site
  • The active site on the ribosome is entirely ribosomal RNA. It catalyzes peptide bond formation, the ribosome is an enzyme called ribozyme
  • the amino acid of the P-site tRNA is transferred to the amino acid on the A site tRNA
  • 1 incoming aminoacyl tRNA, 2 peptide bond formation 3 translocation
• Translocation contin.

  Translocation occurs when the ribosomes slides 1 codon towards the 3' end of the mRNA. Elongation factors help to move the ribosome, energy is needed

  Translocation accomplishes 3 things

  1. the uncharged tRNA from the P site moves into the E site and is ejected from the ribosome
  2. the tRNA attached to the growing protein moves into the P site
  3. Open the A site to expose a new codon, which is now available to accept a new aminoacyl tRNA
  4. the three steps of translocation repeat at each codon along the mRNA
• Terminating Translation

  • Termination occurs when the A site encounters a stop codon
  • A protein call a release factor enters the A site. It resembles tRNAs in size and shape but does not carry an amino acid, also hydrolyzes (breaks) the bond linking the P-site tRNA to the polypeptide chain
  • the newly synthesized polypeptide, tRNAs, and ribosomal subunits separate from the mRNA
  • 1 release factor binds to stop codon, 2 polypeptide and uncharged tRNAs are released 3 ribosome subunits separate
• Overview in Eukaryotes

  1. Transcription- In the nucleus DNA polymerase makes pre-mRNA (primary transcript)
  2. RNA processing- splicing occurs, the 5' Guanine cap and 3' Poly A tail are added, makes mature mRNA
  3. Translation- In cytoplasm the small subunit of the ribosome hold mRNA and uses tRNA
  4. Post translational modification- folding, glycosylation transport, activation, degradation of protein