From DNA to Protein

The Central Dogma

• This is one of the fundamental concepts of molecular biology
• All visible phenotypes (and those you don't see too!) are the result of the actions of enzymes, which are protein catalysts that regulate all body functions
• Any alteration of this process may affect the formation of proteins, hence the expression of the phenotype
• Transcription - the synthesis of RNA under the direction of DNA
• Translation - the actual synthesis of a protein, which occurs under the direction of mRNA

The Players

• DNA - two strands, the coding strand and the template strand
  • The template strand is what is used as a template in the synthesis of mRNA
  • The non-template strand (aka coding strand ) is not used as a template, but is identical in sequence to the mRNA except that all the U's are T's
• RNA - similar to DNA, but less stable (the extra oxygen in the molecule makes it more reactive). Four nucleotides (AUCG) - A=T,. C=G.
• The gene - a series of bases on one strand of DNA which codes for protein
  • Promoter region - region of DNA usually upstream from the gene which regulates gene activity
• RNA polymerase - an enzyme which reads DNA and makes a complementary messenger RNA strand (mRNA) during transcription
• mRNA - the RNA product of transcription
• ribosome - site where the mRNA message is read and protein synthesis takes place
• tRNA - used in synthesis of protein - carries amino acid

Basic Anatomy of a Gene

• Promoter - Region of DNA where RNA polymerase attaches and initiates transcription.  The binding of RNA polymerase to the promoter region of the gene is a very complicated process and will be discussed in future lectures
• Gene - area of DNA which codes mRNA

Transcription

• RNA polymerase is a holoenzyme - a functional enzyme that consists of a core enzyme (w/ active site) and associated proteins
  • Sigma is a detachable protein subunit which must bind to RNA 
• RNA polymerase binds to promoter region and pries the two strands of DNA apart
  • RNA polymerase can only bind to one of the strands of DNA at the 3' end (so that the transcribed mRNA will be constructed 5' to 3')
  • The stretch of DNA that will be transcribed is known as the transcription unit
  • The promoter includes the transcription start point (AUG) and typically extends several dozen bases "upstream"
• RNA polymerase holoenzyme binds to DNA and begins to synthesize mRNA
  • In prokaryotes, RNA polymerase specifically recognizes the promoter and binds
    • Two common promoters are the -10 box and the -35 box (so named because they occur 10 bp and 35 bp upstream from the transcription origin)
    • Sigma binds to the -10 and -35 boxes
    • In prokaryotes, there is a single protein that binds to DNA, but in eukaryotes there are many
  • In eukaryotes, RNA polymerase binding is mediated by one or many proteins called transcription factors
    • These transcription factors must be completely bound to the promoter in order for RNA polymerase to bind
    • The TATA box is a region about 30 bp's up from the start  point which is recognized by the transcription factors
    • The completed assembly of transcription factors and RNA polymerase bound to the promoter is known as the transcription initiation complex
• When RNA polymerase is finished, mRNA leaves and is either modified or immediately used to form protein
  • Transcription in prokaryotes terminates immediately after the stop sequence is transcribed
    • a hairpin forms
  • In eukaryotes, transcription usually proceeds at least 30 bp's after the RNA stop signal.
    • There is usually a AAUAAA sequence transcribed in the mRNA (TTATTT on the DNA).
    • Termination of transcription occurs shortly thereafter

The Genetic Code

• universal - with very few exceptions, all organisms use the same code in the formation of protein
• mRNA bases read as triplets (codons). The codons are non-overlapping
• The genetic code is redundant (64 combinations, only 20 aa's)

• Example: CCC, CCU, CCA, CCG all code for Proline
• Example: AUG CAU UAC UAA
  Codes for: Met---His---Tyr---Stop

A More Detailed Look at tRNA

A tRNA

tRNA is the interpreter of the codons contained in a mRNA The function of tRNA is to transfer amino acids from teh cytoplasm's amino acid pool to a ribosome The Cell keeps its cytoplasm well stocked with all 20 amino acids, either by synthesizing them or by taking them up from the surrounding solution A charged tRNA is one which has a specific amino acid bound to the acceptor arm  Each tRNA has a three-base sequence called the anticodon which binds to a complementary triplet on the mRNA according to the base-pairign rules Wobble - Some Relaxation of Strict Base Pairing There is some "flexibility" in the third position of codon-anticodon base pairing - the third base of a codon is known as the "wobble position" example - the base U of tRNA anticodon can pair with either A or G in the third position of an mRNA codon The most versatile tRNA's are those with Inosine (I), a modified base, in the wobble position I can hydrogen bond with U, C, or A. Wobble explains why the synonymous condons for a given amino acid usually only differ by the the third position

Aminoacyl-tRNA Synthetases

When a ribosome pairs a "CGC" tRNA with "GCG" codon, it expects to find an alanine carried by the tRNA. It has no way of checking; each tRNA is matched with its amino acid long before it reaches the ribosome. The match is made by a collection of remarkable enzymes, the aminoacyl-tRNA synthetases. These enzymes charge each tRNA with the proper amino acid, thus allowing each tRNA to make the proper translation from the genetic code of DNA into the amino acid code of proteins.

Twenty Flavors

Most cells make twenty different aminoacyl-tRNA synthetases, one for each type of amino acid. These twenty enzymes are widely different, each optimized for function with its own particular amino acid and the set of tRNA molecules appropriate to that amino acid.

Surprises from Genome Analyses

Recent analyses of entire genomes revealed a big surprise: some organisms don't have genes for all twenty aminoacyl-tRNA synthetases. They do, however, use all twenty amino acids to construct their proteins. The solution to this paradox revealed, as is often the case in living cells, that more complex mechanisms are used. For instance, some bacteria do not have an enzyme for charging glutamine onto its tRNA. Instead, a single enzyme adds glutamic acid to all of the glutamic acid tRNA molecules and to all of the glutamine tRNA molecules. A second enzyme then converts the glutamic acid into glutamine on the latter tRNA molecules, forming the proper pair.

Ribosomes

• The structure of a ribosome reflects its function of bringing mRNA together with charged tRNAs.
• Each ribosome has thre binding sites for tRNA
  • The P site (peptidyl) - holds the tRNA carrying the growing polypeptide
  • The A site (aminoacyl) - holds the tRNA carrying the next amino acid to be added to the chain
  • The E site (exit) - discharged tRNA's leave the ribosome through this recently discovered site
    • the E site is not shown on the diagrams below on account that the diagrams were made by me before the E site was discovered...yes, I am sooo old

Translation

• The large ribosomal subunit has three sites - P site, A site, E site
  
• Initiation - Ribosome + mRNA + tRNA come together at AUG start codon
  • First, the small sumunit binds to both the mRNA
    • The mRNA will bind to the ribosome binding site (Shine-Dalgarno sequence in bacteria)
    • Initiation factors help this process
  • A special initiator tRNA (bearing a methylated methionine called f-Met) binds to the start sequence
  • Next, the large subunit binds to the small subunit + mRNA + tRNA



• Elongation - tRNA with anticodon comes into A site
  • the mRNA in the A site forms hydrogen bonds with charged tRNA
    • a GTP is required to secure the tRNA into the A site
  • RNA acts as a catalyst and catalyzes the peptide bond formation and breaks the bond between polypeptide chain and tRNA in P site

• Translocation - whole system ratchets down so that the tRNA formerly in the A site is now in the P site

  
  

• The cycle repeats itself until as STOP codon is reached
• Termination - when a stop codon is encountered
  • Release factors bind to codon - there are no tRNA with anticodons to stop factors
  • The release factor causes the addition of a water molecule instead of an amino acid to the polypeptide chain.
  • Whole polypeptide chain falls off
  • The remainder of the translation assembly then comes apart

• This whole process is animated well here

Mutation

• DNA: CCA is changed to TCA
• Then RNA: GGU [Gly] is changed to AGU [Ser]

• DNA: GAT is changed to GAA
• RNA: CUA [Leu] changed to CUU [Leu]

Neutral (silent) mutation - a base pair changes, but there is no change in the protein
Missense mutation - A mutation that alters a codon for a particular amino acid to one specifying a different amino acid
Nonsense mutation - A point mutation that causes the change of an amino acid codon into a stop codon
Frameshift mutation - A mutation that alters the normal triplet reading frame so that codons downstream from the mutation are out of register and not read properly.

1.  Identify a molecule as either DNA or RNA
2.  Create a complementary strand of DNA given the template strand
3.  Given a piece of DNA (coding or template), generate the protein that is coded in the DNA
4.  Given a piece of mRNA, translate into protein
5.  Determine how a given mutation will affect the protein