1. gene expression… from DNA to protein
biology 1

2. Genes control metabolism Gene expression is a two stage process
transcription
translation
Genes consists of triplets of nucleotides - the genetic code
Protein synthesis in prokaryotes and eukaryotes
Eukaryotic modification of RNA
Mutations

3. Genes control metabolism
One gene-One polypeptide rule
Polypeptides that are constructed as a result of transcription/translation process become either
structural proteins
enzymes
Those proteins that have quaternary structure may have polypeptides originating from different genes

4. The transcription/translation process
Transcription: DNA codes for the construction of mRNA
Translation: mRNA is read by rRNA at a ribosome; tRNA brings amino acids to ribosome as defined by code on mRNA
Ribosome assembles polypeptide
Recap on RNA - a ribose nucleic acid that uses Uracil (U) in place of Thymine (T)

5. The genetic code
The linear sequence of nucleotides in DNA ultimately determines the linear sequence of amino acids in a polypeptide
There are approximately 20 types of amino acid to choose from
In DNA, the four nucleotides are ATCG
Therefore, the sequence of four possible nucleotides must code for 20 amino acids
If DNA used a individual nucleotide to refer to an individual amino acid, this system would only code for 41 amino acids
Using two nucleotides would account for 42 = 16 amino acids
Using three nucleotides would account for 43 = 64 amino acids
Since there are only 20 amino acids, yet 64 possible codes, some redundancy occurs

6. Each block of three nucleotides, ultimately corresponding to a particular amino acid, is called a codon
In the first stage of the gene expression process, transcription, the information in the codons of a gene are transferred to mRNA
This process is via an RNA polymerase that uses one of the DNA strands of the double helix (the template strand)
For each amino acid, there are generally several codons possible. Also, some codons have a non-amino acid equivalent, but instead send specific messages to RNA polymerase (start/stop)

7. Transcription Three phases
Polymerase binding and initiation
Elongation
Termination
In eukaryotes, RNA polymerase II bind to specific regions on DNA called promoters
Promoters are typically 100 nucleotides long, including
The initiation site, where transcription begins
Nucleotides sequences that help initiate transcription

8. Initiation in eukaryotes requires transcription factors, DNA-binding proteins that bind to specific nucleotide sequences in the promoter region
A common place for a transcription factor to bind is the TATA box
RNA polymerase recognizes the promoter site once DNA and transcription factor have bound at the TATA box
RNA polymerase temporarily separates the double helix for transcription

9. In elongation, RNA polymerase II (eukaryotes)
Untwists the DNA molecule
Adds incoming RNA free-floating nucleotides to the 3’ end of the RNA strand (grows 5’ to 3’)
mRNA grows at nucleotides/sec. The mRNA chain starts to peel away as the double helix reforms
Followed in series, several molecules of RNA polymerase can simultaneously transcribe the same gene
Transcription proceeds until the polymerase reaches a termination code

10. Translation
During translation, proteins are synthesized according to a genetic message of sequential codons along mRNA
tRNA (transfer RNA) interprets between the base sequence in mRNA and the amino acid sequence in a polypeptide chain. To do this…
Transfer amino acids from cytoplasm to ribosome
Recognize the correct codons on mRNA
Molecules of tRNA are specific to one particular amino acid
One end of tRNA attaches to a specific amino acid (3’ end)
The other end attaches to an mRNA codon by base pairing with its anti-codon

11. An anti-codon is a nucleotide triplet in tRNA
tRNA decodes the genetic message codon by codon
There are 45 types of tRNA, which is sufficient for the 64 codes, since there is a relaxation of base-pairing on the third nucleotide (wobble)
e.g., U in 3rd position of anticodon can bind with A or G on the equivalent codon
In some cases, third position on a tRNA anticodon is occupied by Inosine (a sixth nucleotide) that can bind with U, C or A

12. Each amino acid has a particular synthetase enzyme
Joining of tRNA to specific amino acid at the 3’ end is by Aminoacyl-tRNA synthetase
Each amino acid has a particular synthetase enzyme
ATP activates the amino acid by losing 2 phosphate groups, and joining to the amino acid as AMP
tRNA bonds to the amino acid, which loses AMP
Ribosomes coordinate the pairing of tRNA anticodons to mRNA codons
Consist of 2 subunits (small and large) that remain separated when not involved in protein synthesis
Ribosomes are composed of 60% rRNA and 40% protein

13. Building of a polypeptide chain consists of three steps
In addition to an mRNA binding site, two further sites on a ribosome are the P- and A-sites
P-site holds the tRNA carrying the growing polypeptide chain
A-site holds the tRNA that has the next amino acid in the polypeptide sequence
Building of a polypeptide chain consists of three steps
Initiation
Elongation
Termination

14. Translation Initiation
In eukaryotes, the small ribosomal unit binds to an initiator tRNA (methionine; anticodon UAC)
The small ribosomal unit binds to the 5’ end of mRNA, and in doing so brings the tRNA anticodon in close proximity with mRNA methionine codon
This binding requires initiation factors
Finally, the large subunit binds to the complex
The initiator tRNA fits to the p-site of the ribosome
The vacant a-site is ready for the next aminoacyl-tRNA complex

15. Translation elongation
Codon recognition—mRNA codon in the a-site of the ribosome forms hydrogen bonds with anti-codon of an entering tRNA carrying the next amino acid in the chain
Peptide bond formation—The enzyme peptidyl transferase (part of the large ribosomal unit) catalyzes the peptide bond between the incoming amino acid and the growing polypeptide chain
Translocation—the tRNA in the p-site releases from the ribosome, and the tRNA in the a-site moves into the vacated site

16. Translation termination
A termination codon signals the end of translation; by binding to a protein release factor, this causes:
Peptidyl transferase hydrolyzes the bond between the completed polypeptide and the tRNA in the p-site
This frees the polypeptide and tRNA so that they can release from the ribosome
The two ribosomal units disassociate
mRNA may continue to be translated by polyribosomes

17. Differences between prokaryotic and eukaryotic gene expression
Lack of nuclear membrane in prokaryotes means that transcription can occur at one end of the mRNA molecule, while translation can be occurring at the other end
In eukaryotes, RNA is modified following transcription before translation
5’ cap added (modified guanine nucleotide
Poly-A tail added (200 adenine nucleotides) to 3’ end
These ends might protect mRNA sequence (attaching to untranslated leader and trailer sequences respectively)
Gene splicing

18. Gene splicing
Eukaryotic mRNA has segments of non-code, called introns (code sequences called exons)
Introns and exons are initially coded into one long strand called hnRNA (heterogenous RNA)
In RNA splicing, introns are removed from hnRNA to make mRNA
Process of splicing mRNA involves SnRNPs (“snurps”) - small nuclear ribonucleoproteins, that are composed of SnRNA (small nuclear RNA) and proteins
Together with extra proteins, SnRNPs form complexes called spliceosomes, which excise introns (SnRNPs attach to either end of each intron)
tRNA and rRNA also need to be spliced, but different agents do the splicing - ribozymes, RNA molecules that act as enzymes (note: thus not all enzymes are proteins)

19. Why do introns exist? May regulate gene activity
Splicing may regulate export of mRNA to cytoplasm
Introns cause exons to be further apart, and therefore to be further away from each other on the chromosome: this could mean a higher probability of recombination during cross-over
Specific introns may code for specific domains within a protein

20. When things go wrong... MUTATION!
Mutation = a permanent change in DNA that can involve large chromosomal regions or a single nucleotide pair
Point mutation = a mutation limited to one or two nucleotides in a single gene
Base-pair substitution
Missense mutation
Nonsense mutation
Insertion/deletion mutations

21. Base-pair substitutions generally have no effect if they occur on the third nucleotide of a triplet
If they do change the amino acid, one a.a. substitution may not radically affect the functionality of the final polypeptide
In some cases, functionality is improved: in most cases, functionality is impaired
In nonsense mutations, the substitutions causes a triplet to read STOP, abruptly terminating polypeptide chain. Such mutations are usually harmful

22. Insertions or deletions add or remove one or more nucleotides from a sequence
Since a reading frame for nucleotides is based on a series of three, insertions and deletions that add or remove a sequence of nucleotides not divisible by 3 can substantially alter the final polypeptide
Such a mutation is referred to as a frameshift - these mutations usually result in non-functional proteins, unless they occur towards the end of a sequence