3. How do genes produce proteins?
Gene expression: the process by which genes
produce proteins
Two stages:
• Transcription
• Translation
4. Steps of transcription
1. Initiation need two important parts;
promotor sequence and Initiation factors or transcription factors
2. Elongation
3. Transcription termination
6. • Basically, elongation is the stage when the RNA strand gets
longer, thanks to the addition of new nucleotides.
2. Elongation
• RNA polymerase "walks" along one
strand of DNA, known as the template
strand (3' to 5' direction)
• RNA polymerase adds a
matching (complementary)
RNA nucleotide to the 3' end of
the RNA strand.
• The RNA transcript is nearly
identical to the non-template, or
coding, strand of DNA.
7. 3. Transcription termination
• The process of ending transcription is called termination, and it
happens once the polymerase transcribes a sequence of DNA
known as a terminator
• Termination in bacteria
There are two major termination strategies found in bacteria:
• Rho-dependent
• Rho-independent.
8. In eukaryotes:
Very little is known about how they terminate
transcription, but termination may occur by a
special signalling of transcription factors
themselves.
9. Post - transcriptional modification
• The molecule that's directly made by transcription in one of your
(eukaryotic) cells is called a pre-mRNA
1. Addition of a 5' cap to the beginning of the RNA
2. Addition of a poly-A tail (tail of A nucleotides) to the end of the RNA
3. (RNA splicing) Chopping out of introns, or "junk" sequences, and
pasting together of the remaining, good sequences (exons)
10. • Both the cap and the tail protect the transcript and help it
get exported from the nucleus and translated on the
ribosomes (protein-making "machines") found in the
cytosol.
• The cap is a modified guanine (G) nucleotide, and it protects the
transcript from being broken down. It also helps the ribosome attach
to the mRNA and start reading it to make a protein.
• a sequence called a polyadenylation signal activates another enzyme adds about
100 - 200 adenine (A) nucleotides to the cut end, forming a poly-A tail. The tail
makes the transcript more stable and helps it get exported from the nucleus to
the cytosol.
11. • Translation is the process by which a protein is
synthesized from the information contained in a molecule
of messenger RNA (mRNA). During translation, a mRNA
sequence is read using the genetic code, which is a set of
rules that defines how an mRNA sequence is to be
translated into the 20-letter code of amino acids, which
are the building blocks of proteins.
Translation / RNA translation
12. The genetic code
• In an mRNA, the instructions for building a polypeptide are RNA nucleotides (As,
Us, Cs, and Gs) read in groups of three. These groups of three are called codons.
• The genetic code is nearly universal, shared by the simplest bacteria to the most
complex animals.
There are 64 codons, but only 20 amino acids
• One amino acid will have multiple codons
• Each codon specifies the amino acid (one of 20) to be placed at the corresponding
position along a polypeptide
• One codon, AUG, specifies the amino acid methionine and also acts as a start
codon to signal the start of protein construction.
• There are three more codons that do not specify amino acids. These stop codons, UAA,
UAG, and UGA, tell the cell when a polypeptide is complete
• Codons along an mRNA molecule are read by translation machinery in the 5' to 3' direction
14. The players are;
• tRNA
• Aminoacyl-tRNA synthetase
• Ribosomes
• Amino acids
• Chaperone proteins
Overview of translation
15. Specific tRNAs will attach to specific amino acids
• How does it ‘know’ which amino acid to attach to?
– The anticodon on the end dictates which amino acid the tRNA will attach to
– tRNAs don’t naturally come attached to their amino acids
– An enzyme called aminoacyl-tRNA synthetase catalyzes the linking of an amino
acid to a tRNA
16.
17. • Roughly the same for both eukaryotes and prokaryotes
• Ribosomes provide a site for tRNA and mRNA to come together
• Composed of rRNA and proteins
Two populations of ribosomes in cells: free ribosomes (in the cytosol)
and bound ribosomes (attached to the ER). Free ribosomes mostly
synthesize proteins that function in the cytosol. Bound ribosomes
make proteins of the endomembrane system and proteins that are
secreted from the cell.
– Large subunit and small subunit
– Three catalytic sites: E, P, and A
• “Aminoacyl-tRNA binding site”
• “Peptidyl-tRNA binding site”
• “Exit site”
Ribosomes
18.
19. 1. Initiation ("beginning"): in this stage, the
ribosome gets together with the mRNA and the first
tRNA so translation can begin.
2. Elongation ("middle"): in this stage, amino acids
are brought to the ribosome by tRNAs and linked
together to form a chain.
3. Termination ("end"): in the last stage, the
finished polypeptide is released to go and do its job
in the cell.
The steps of translation
20. In order for translation to start, we need a few key
ingredients. These include:
1. A ribosome (which comes in two pieces, large and small)
2. An mRNA with instructions for the protein we'll build
3. An "initiator" tRNA carrying the first amino acid in the
protein, which is almost always methionine (Met)
Initiation
21. • During initiation, these pieces must come together in just the right
way. Together, they form the initiation complex, the molecular
setup needed to start making a new protein.
• Initiation depends on specialized protein "helpers" called initiation
factors. Their job is to help the ribosome subunits, tRNA, and mRNA
find each other in an orderly and predictable way.
• Also, initiation ingredients around requires energy. The energy is
provided by the cell in the form of guanosine triphosphate (GTP), a
common "energy currency" molecule that's similar to the ATP.
22. Inside cells, translation initiation goes like this:
first, the tRNA carrying methionine attaches to the small ribosomal
subunit. Together, they bind to the 5' end of the mRNA by recognizing
the 5' GTP cap (added during processing in the nucleus). Then, they
"walk" along the mRNA in the 3' direction, stopping when they reach
the start codon. Large ribosomal subunit binds through GTP hydrolysis
(energy provider) and begins elongation.
23. Amino acids are added one by one to the preceding
amino acid at the C-terminus of the growing chain.
Each addition involves proteins called elongation
factors and occurs in three steps:
1. codon recognition,
2. peptide bond formation,
3. translocation
Elongation
24. 1. Codon recognition
– Three ribonucleotides are exposed in the “A” site of ribosome. A
tRNA with the correct anticodon will recognize the exposed codon
and enter the “A” site.
2. Peptide bond formation
– A peptide bond is formed between the two amino acids of adjacent
tRNAs on the “A” and “P” sites.
3. Translocation
– The ribosome moves one codon forward, the tRNA now in the “E”
site exits and a new codon is now exposed in the “A” site.
25.
26. • The codons UGA, UAA, and UAG are stop signals,
instead of a tRNA binding there; a protein called a release
factor binds and causes the ribosome to release.
• The release factor causes the addition of a water
molecule instead of an amino acid.
• This reaction releases the polypeptide, and the
translation assembly then comes apart
Termination
27. Polyribosomes:
Many ribosomes can attach to one mRNA at once.
This allows for very fast translation of multiple copies of the protein.
28. Once mRNA translated, proteins aren’t finished! They must
undergo post-translational modification that involved
important functions such as;
1. Help with proper folding
2. Addition of functional groups
3. Building subunits together into a protein with quaternary
structure
4. Cleaving apart a protein to activate it
Post-translational modifications