DNA -> mRNA
- Occurs in the nucleus (in eukaryotes)
- Makes a single stranded copy of mRNA that can leave the nucleus
3 steps of transcription
Initiation in transcription
Transcriptions factors and RNA polymerase bind to the promoter of the gene (which genes are transcribed is tightly controlled)
Elongation in transcription
- DNA is unwound and RNA polymerase creates a strand of mRNA which is complementary to the template strand of DNA
- RNA polymerase synthesizes the mRNA in the 5' to 3' direction
- Occurs at a rate of ~20 bases per second
Termination in transcription
Transcription proceeds until the polymerase reaches the terminator sequence which causes the enzyme to dissociate from the DNA
Initiation in bacteria
- RNA polymerase + sigma (holoenzyme) can recognize and bind to promoter
- -Different sigma proteins bind promoters with different DNA sequences
RNA polymerase in elongation
- Unwinds the helix and exposes 10-20 nucleotides at a time
- Synthesizes mRNA from 5'->3'
- --Does not need primer
- Rate= 50 nucleotides per second
Termination in bacteria
- Transcription proceeds through a termination sequence
- RNA forms hairpin
- RNA polymerase releases transcript
Differences in eukaryotic transcription
- 3 main polymerases (each produces a certain type of RNA)
- Promoters more divers (TATA box)
- Basal transcription factors (not sigma) help RNA polymerase to recognize the promoter
- Termination [poly(A) signal- signals the end of transcription]
- Transcription and translation separated
DNA template strand 5'_________3'
Complementary strand 3'_________5'
In the transcription, where would the promoter be located?
- C) to the right of the template strand
- RNA is synthesized 5->3, so it gets read from the other way
- Reads from 3'->5', so new bases will be added 5'->3'
The initial transcript in RNA processing in Eukaryotes
- Must be modified in the nucleus before it can be transported to the cytoplasm for translation
- -5' cap= modified guanine added to 5' end
- -3' poly-A tail= enzyme adds 50-250 adenines to 3' end
Important functions of pre-mRNA
- Facilitate export from nucleus
- Protect from degradation in cytoplasm
- Help ribosomes attach to the 5' end for translation
- Long stretches of noncoding DNA in most eukaryotic genes
- Between exons
Coding segments in most eukaryotic genes
What must happen to introns in RNA splicing
The intervening sequences (introns) are transcribed, but must be removed before translation
- snRNAs + proteins= snRNPs (small nuclear ribonucleoproteins)
- -recognize specific sequences at the end of introns
- Cuts out introns and joins the exons back together
5' UTR EI I1 E2 I2 E3 I3 E4 UTR 3'
Which components of the previous molecule will also be found in mRNA in the cytosol?
B) 5' UTR E1 E2 E3 E4 UTR 3'
Alternative RNA splicing...
B) can allow the production of proteins of different sizes and functions from a single mRNA
Why do we have introns?
- Some introns contain sequences which play a role in regulating gene expression
- Allows a single gene to code for more than one polypeptide
- They let you take out or keep exons
Alternative RNA splicing
- Some exons can be left out of final transcript
- This allows humans to make 75,000-100,000 proteins with only ~20,000 genes
mRNA -> protein
- Occurs in the cytoplasm
- mRNA is "read" 3 bases at a time (codon) to build a polypeptide
Key players of translation
The codon for serine is UCG. The coding (non-template) strand of the DNA that encoded is ___ and the anticodon of the tRNA that recognizes it is ___.
B) TCG, AGC
Transfer RNA (tRNA)
- Translates messages from nucleic acids to amino acids
- Transcribed from DNA template and folds to form 3D structure
- Contains an anticodon
- Carries a specific amino acid
Triplet which is complementary to codon on mRNA
During eukaryotic translation, which of the following is NOT used?
D) RNA polymerase
- Enzymes which attach the amino acid to the tRNA
- Active site can only fit specific combinations of tRNA and amino acids (ensures anticodon brings in correct amino acid)
- tRNA with amino acid attached is "charged"
- Only ~40 tRNAs (not the 61 you would expect... 1 for each possible codon)
- --Flexible base pairing in 3rd position = wobble
How many different aminoacyl-tRNA synthetases are there?
20 different synthetases (1 for each amino acid)
A mutant bacterial cell has a defective aminacyl-tRNA synthetase that attaches a lysine to tRNAs with the anticodon AAA instead of the normal phenylalane. The consequence for the cell will be that...
B) Proteins in the cell will include lysine instead of phenylalanine at amino acid positions specified by the codon UUU
onsist of a small and large subunit composed of protein and ribosomal RNA (rRNA)
Why are prokaryotic and eukaryotic ribosomes differences important in medicine?
- Some antibiotic inactivate bacterial ribosomes without harming our own
- Otherwise, the two ribosomes are very similar
What catalyzes the formation of peptide bonds?
rRNA not the protein
3 steps of translation
Initiation in translation
- Small unit of the ribosome, which the initiator tRNA (methione) already attached, binds to leader sequence of mRNA and scans for AUG
- Large subunit binds and completes translation initiation complex
Elongation in translation
Ribosome moves along mRNA adding amino acids to the growing polypeptide chain
Termination in translation
Ribosome hits a stop codon and the subunits separate the polypeptide is released
Initiation process in translation
- rRNA in small subunit binds to complementary sequence on mRNA (ribosomal binding site)
- --helped by initiation factors
- Initiator tRNA binds to start codon
- Large subunit attaches to form translation initiation complex
Elongation process in translation
- Amino acids are added one by one to growing polypeptide chain
- -Requires proteins= elongation factors
- -Requires energy
3 ribosome sites in elongation
- E- empty tRNA exits
- P- tRNA with growing polypeptide
- A- incoming (active) tRNA
Termination process in translation
- Elongation continues until a stop codon is reached
- A release factor (protein shaped like a tRNA) binds to the stop codon in the A site
- -causes the polypeptide to be released
- The translation assembly breaks apart
- Typically a single mRNA is simultaneously translated by multiple ribosomes (polyribosomes)
- 1 ribosome can make an average polypeptide in less than a minute
- Polypeptide must undergo correct secondary and tertiary folding
- -may require assistance of molecular chaperons
Chemical post-translational modifications
- Attachment of sugars, lipids, phosphate groups
- Cleavage of polypeptide chain (i.e insulin)
- Interaction of multiple subunits (quaternary structure)