unit 4

• DNA serves as a template for the synthesis of an RNA molecule, which then directs the synthesis of a protein product

  • Sometimes, the RNA itself is the final product
  • The principle of directional information flow from DNA to RNA to protein is the central dogma of molecule biology
• transcription: RNA synthesis using DNA as a template
• translation: synthesis of protein using the information in the RNA
• Mutations that alter sequences near the 5' end of mRNA result in alterations near the corresponding protein's N-terminal end. whereas mutations that alter the 3' sequences of mRNA result in alterations in the protein's C-terminal end. What do these findings imply?


  The order of nucleotides from 5' to 3' in mRNA determines the order of amino acids from N- to C-termini
• Transcription and translation involve many of the same components in bacteria and eukaryotes

  • Messenger RNA, mRNA, is RNA that is translated into protein
  • Ribosomal RNA, rRNA, is an integral component of the ribosome
  • Transfer RNA, tRNA, molecules serve as intermediaries bring amino acids to the ribosome
  • The latter two functions in translation
• All three major classes of RNA are

  (a) synthesized by transcription of the appropriate DNA sequences

  (b) involved in the process of translation. the appropriate amino acids are brought to the mRNA and ribosome by tRNA. A tRNA molecule carrying an amino acid is called aminoacyl tRNA.

  (c) polypeptides then fold and assemble into functional proteins

  
• The Genetic Code

  • The relationship between the DNA base sequence and the linear order of amino acids in the protein products is based on a set of rules known as the genetic code
  • There are four DNA bases and 20 amino acids
  • A doublet code, in which two bases specify a single amino acid, is inadequate because only 16 combinations are possible
  • A triplet code, in which combinations of three bases specify amino acid, would have 64 possible combinations, more than enough for all 20 amino acids
• Supporting a triplet code using frameshift mutations

  • inserting or deleting a nucleotide (indel mutations) causes the rest of the sequence to be read out of phase -- this is a shift in the reading frame
  • mutations that cause insertion or deletion of a nucleotide are thus called frameshift mutations
  • proflavin is a mutagen that induces insertion or deletion of single nucelotides
  • experiments using Proflavin supported the idea of a triplet code
• the interpretation of revertible mutations

  • no revertant single mutants
  • revertant double mutations if the two mutations are opposite
  • Revertant triple mutants only if all mutations are the same

  
• The Genetic Code is Degenerate and Nonoverlapping

  • There are 64 combinations of nucleotide triplets and only 20 amino acids
  • This means the genetic code is a degenerate code, meaning that a particular amino acid can be specified by more than one triplet
  • it is also nonoverlapping; the reading frame advances three nucleotides at a time

  
• Degeneracy


  Enhances the adaptability of the coding system
• If only 20 triplets were assigned a coding function, any mutation in DNA that lead to the formation of any of the other 44 possible triplets would interrupt the genetic message at the point
• Overlapping genetic code


  The insertion or deletion of a single base pair in the gene would lead to the insertion or deletion of one amino acid at one point in the polypeptide and would change several adjacent amino acids, but if would not affect the reading frame of the remainder of the gene
• If the genetic code were overlapping, there would be no observed frame-shift mutations
• The codon dictionary was established using synthetic RNA polymers and triplets

  • RNA triplets, called codons, are read by the. transcriptional machinery
  • further homopolymer experiments showed AAA codes for lysine, and CCC codes for proline
  • as synthetic polymer technology progressed, production of all different codons independently led to the elucidation of the entire codon dictionary
• Messenger RNA guides the synthesis of polypeptide chains

  • mRNA is transcribed from DNA similarly to how DNA is replicated, but with two differences
  • in mRNA synthesis, only one DNA strand is copied, called the template strand; the other strand is called the coding strand because it is similar to the mRNA sequence
  • in mRNA synthesis, a uracil base (U) is used instead of thymine
• Transcription


  1. Binding

  2. Initiation

  3. Elongation

  4. Termination
• Transcription unit


  The DNA that gives rise to one RNA molecule
• Transcription initiation


  1. RNA polymerase binds to a promoter sequence

  2. Triggers local unwinding of the double helix
• Transcription elongation


  1. RNA polymerase moves along the DNA template

  2. Unwinds the helix

  3. Elongates the RNA
• Transcription termination


  1. RNA polymerase dissociates from the DNA template

  2. Synthesis is terminated

  3. RNA molecule is released
• • RNA polymerase binds to a DNA promoter site, a sequence of several dozen base pairs that determines where RNA synthesis will start
  • The terms upstream and downstream refer to sequences located toward the 5' or 3' end of the transcription unit, respectively
• Overview of transcription

  1. binding of RNA polymerase to DNA at a promoter
  2. initiation of transcription on the template DNA strand
  3. subsequent elongation of the RNA chain
  4. eventual termination of transcription, accompanied by the release of RNA polymerase and the completed RNA product from the DNA template

  
• RNA polymerase moves along the template strand of the DNA in the 3' -> 5', and the RNA molecule grows in the 5' -> 3'
• Essential sequences in a typical bacterial promoter

  • the transcription start site is almost always a purine and usually an adenine
  • about 10bp upstream of the start site is the sequence TATAAT, called the 10 sequence of the Pribnow box
  • At or near the -35 position is the sequence TTGACA called the -35 sequence
• Organization of a bacterial promoter sequence

  • the promoter region in bacteria is a stretch of about 40bp adjacent to and including the transcription start site.
  • By convention, the critical DNA sequences are given as they appear on the coding strand.
  • essential features of the promoter are the start site (designated +1 and usually an A), the six-nucleotide -10 sequence, and the six-nucleotide -35 sequence.

  
• Transcriptional initiation in bacteria

  1. RNA polymerase binds to -35 and -10 sequences in the promoter via the subunit. An additional upstream (UP) element may be bound by the alpha subunits
  2. while tightly bound to the promoter, the polymerase pulls additional DNA toward itself
  3. eventually, a sufficiently long RNA is produced that the polymerase escapes the promoter, releasing the subunit

  
• Elongation of the RNA chain

  • Chain elongation continues as RNA polymerase moves along the DNA molecule
  • The RNA is elongated in the 5' to 3' direction, with each new nucleotide added to the 3' end
  • As the polymerase moves along the DNA strand, the double helix ahead of the polymerase is unwound, and the DNA behind it is rewound into a double helix
• During elongation, RNA polymerase binds to about 30bp of DNA. At any given moment, about 18bp of DNA are unwound, and the most recently synthesized RNA is still hydrogen-bonded to the DNA, forming a short RNA-DNA hybrid about 8-9bp long. The total length of growing RNA bound to the enzyme and/or DNA is about 25 nucleotieds

  
• Termination of RNA Synthesis

  • elongation of the RNA chain proceeds until the RNA polymerase copies a sequence called the termination signal
  • many termination sequences contain a short GC-rich sequence followed by several U's
  • The GC region in the RNA forms a hairpin loop pulling the RNA molecule away from the DNA
  • The the bonds between the U's and the A's of the template strand break, releasing the RNA

  
• Transcription in Eukaryotic Cells has additional complexity Compared with Prokaryotes

  • Eukaryotic transcription involves the same four stages as prokaryotic, but there are several important differences
  • Three different RNA polymerases transcribe one or more different classes of RNA
  • Eukaryotic promoters are more varied than bacterial ones; some are even located downstream of the gene
• Eukaryotic transcription

  • eukaryotic transcription differs from that of prokaryotes
  • RNA polymerases in eukaryotes require additional proteins called transcription factors, some of which must bind before the RNA polymerase can bind
  • Protein-protein interactions play a prominent role in eukaryotic transcription
• • RNA cleavage is more important than termination of transcription in determining the 3' end of the transcript
  • Newly forming RNA molecules undergo RNA processing, chemical modification during and after transcription
• RNA polymerase I, II and III carry out transcription in the eukaryotic nucleus

  • there are three RNA polymerases in the nucleus, designated RNA polymerases I, II, and III

  
• Three classes of promoters are found in eukaryotic nuclear genes, one for each type of RNA polymerase

  • the core promoter is the smallest set of DNA sequences that initiates transcription
• The promoter for RNA Polymerase II

  • At least four types of DNA sequences are involved in core promoter function

  1. A short initiator sequence surrounds the transcription start point
  2. the TATA box, a consensus sequence of TATA followed by two to three A's, is located about 25 nucleotides upstream of the start point
  3. The TFIIB recognition element (BRE) is located slightly upstream of the TATA box
  4. The down stream promoter element (DPE) is located about 30 nucleotides downstream from the start point
• TATA-driven promoters contain

  • TATA box
  • INR
• TATA-driven promoters contain

  • TATA box
  • INR
• TATA-driven promoter

  • TATA box
  • INR

  May or may not contain

  • BRE

  No DPE sequence
• DPE-driven promoters contain

  • DPE sequence
  • INR

  No TATA box

  No BRE