Lecture 25 Flashcards
Central dogma of molecular biology
DNA (dna replication)
to RNA through transcription
To proteins through translation
Gene expression
Genes encode for messenger RNA (mRNA) which code for proteins (central dogma)
Genes encode for functional RNAs such as transfer RNAs (tRNA) and rinosomal RNAs (rRNA)
Generally activity of proteins produce the inherited traits of organism
Can flow of genetic information be reversed?
No
Unless it’s an RNA virus whose rna can be used to code for DNA in a host cell (these viruses have the enzyme reverse transcriptase)
Gene expression is the process by which
Information from a gene is used in the synthesis of a functional gene product
Gene products
Often coded for by messenger RNAs /mRNAs but they can also be functional RNAs such as transfer RNAs (tRNAs) or ribosomal RNAs (rRNAs)
Transcriptome
Totality of RNA transcripts expressed by a cell or an organism
Proteome
Entire set of proteins that is/ or can be expressed by a genome, cell, tissue, or organism at a certain time
Transcription
Synthesis of RNA molecule complementary to a portion of a strand of DNA acting as a template
Thousands of transcripts are being produced every second in cells
Occurs in 3 stages
Occurs in the nucleus in eukaryotes and cytoplasm in prokaryotes
3 transcription stages
Initiation
Elongation
Termination
Where does transcription occur in a eukaryote and prokaryote
Eukaryote- nucleus
Prokaryote- cytoplasm
Gene consists of what? In terms of transcription
Promoter and a transcription unit
Specific sequences of nucleotide in prokaryote and eukaryote promoters differ
Eukaryotes have a TATA box within their promoter consisting of 30 bases pairs of Ts and As located upstream from the transcription start point
Transcription unit
Stretch of DNA that transcribes a primary transcript (the RNA molecule)
Initiation steps
Transcription factor proteins bind to the promoter in the areas of the TATA box
Transcription factors recruit RNA polymerase.
- combination of transcription factors and RNA polymerase forms the transcription initiation complex
- only 1 of the strands to be transcribed is the template strand
Elongation steps
As RNA polymerase slides along the transcription unit, it separates the DNA strands and opens the transcription bubble
RNA polymerase breaks the H bonds between the nitrogenous bases of the 2 DNA strands
This allows in of the DNA strands to function as a template for RNA synthesis
RNA is transcribed via complementary base pairing (just like dna except it’s AU/CG in RNA)
RNA transcribed from 5 to 3’ ends adding new RNA nucleosides to the 3’ end. Using triphosphate ribonucleosides
DNA left behind by RNA polymerase recoils back into double helix (note: newly synthesized RNA molecules consist of a single strand. Not a double helix)
4 different ribonucleoside triphosphates used to form RNA molecule
Contain a 5 carbon ribose sugar instead of a deoxyribose sugar that is used in DNA nucleotides (note the hydroxyl OH group on the 2’ carbon)
This means an A encountered on the DNA template strand with result in uracil (U) nucleotide added to the growing RNA molecule
Instead of Thymine
Uracil is a pyrimidine like thymine
Which RNA sequence below is complementary to the given DNA sequence 5’ TTAAGGCC3’
Because RNA transcription is antiparallel, we rewrite the DNA sequence to
3’CCGGAATT5’
Then follow the AC/TG rule
5’GGCCUUAA3’
Termination
When RNA polymerase reaches the end of a gene it encounters a terminator sequence of nucleotides
This signals termination of transcription
RNA polymerase and the RNA transcript are released from the DNA
Recap
DNA to RNA
initiation to
Elongation to
Termination
mRNA
A long polynucleotide strand possessing the code for a particular polypeptide chain on it
mRNA code
Only 4 letters
A. G. C. U
How many amino acids and what are they
Building blocks of protein
20 amino acids
mRNA codes of length 1,2,3 could code for
4 amino acids
Length 1 4^1=4
Length 2 4^2 =16
Length 3. 4^3=64
Codons
3 letter words (ex. 3 nucleotide bases in a row) are called codons
As we saw this means there are 4^3 =64 possible codons that make up the genetic code
Genetic code
Start codon (AUG) starts translation but also encodes for amino acid methionine
Start codon
AUG
Typically
Stop codons examples
UAA
UAG
UGA
Stop codons
Stop translation
Don’t code for any amino acids
When can bacterial mRNAs be translated
They can be immediately translated into polypeptides once they are transcribed in the cytoplasm
Where are eukaryote mRNAs synthesized
In the nucleus and have to be processed or packaged for release into cytoplasm
Eukaryotic packaging
Initial mRNA transcribed in the nucleus is called pre-mRNA (precursor mRNA)
After processing in nucleus RNAs are called mature mRNAs
RNA processing involves 3 steps
Additional of a guanine cap
Addition of a poly (A) tail
Removal of introns by splicing
Addition of a guanine cap stage in RNA processing
A guanine (or methylated) cap is added to 5’ end of a pre-mRNA
by a capping enzyme while the mRNA is still being transcribed in the nucleus
- protects mRNAs from degradation by RNA exonucleases once the mRNAs enter the cytosol
- facilitates the transport of mRNAs from the nucleus, out through the nuclear pores, and into the cytosol
- is the site where ribosomes attach to mRNA at the beginning of translation in the cytosol
Addition of poly (A) tail
Stage in RNA processing
Polyadenylation signal allows enzyme poly(A) polymerase in the nucleus to attach to pre-mRNAs and add 50-250 adenine nucleotides one at a time to the 3’ end of the pre-mRNAs
This forms a poly(A) tail at the 3’ end of each pre-mRNA
Why is the poly (A) tail important in RNA processing
To allow for export through nuclear membrane
Makes translation of mRNA into amino acids and proteins easier
Length of the tail may determine the number of times an mRNA is translated before it is enzymatically degraded in the cytosol by RNA exonucleases.
Removing of introns by splicing in translation of RNA
Pre-mRNAs contain alternating sections of introns and exons
Introns are spliced out of the mRNA and the exons are connected together such that mRNA includes only exons
Therefore all introns are excluded from mature mRNAs
Exons
Expressed sequences
Exit nucleus
RNA processing
Introns
Intervening sequences
Staying in the INterior of the nucleus
In RNA processing
Summary of packaging RNA
Transcription->
Addition of poly (A) tail and RNA splicing ->
Mature mRNA
mRNA mature
Consists of a protein coding region, 2 untranslated regions (UTRs), 5’ cap and a 3’ poly(A) tail
Transfer RNA (tRNA)
No affinity between amino acids and mRNAs, so u need something that can connect them
Like a code reader
tRNAs are the code readers
Small RNAs made in the nucleus (74-95 nucleotides in length) that are highly folded. Having both secondary and tertiary structure in addition to their primary structures
Primary structure
Sequence of nucleotides making up the RNA molecule
tRNA secondary structure
Like a clover leaf with 3 stem loops where they are double stranded due to complementary base pairing
At one end of the tRNA there is a site that can associate with an amino acid
Complementary base pairing between nucleotides in a stem loop
At the end is a region of 3 nucleotide called an anticodon that is complementary to one of the mRNA codons
Add amino acids to the tRNA
Aminoacyl-tRNA synthetase enzymes in the cytosol add the correct amino acid into the tRNA with the appropriate anticodon
Translation
Synthesis of a polypeptide on a ribosome using the code from an mRNA (translated from DNA)
Occurs in cytosol in both eukaryotes and prokaryotes
3 stages. Initiation, elongation, termination
Where does translation occur
In prokaryotes? Eukaryotes?
Cytosol for both
3 stages of translation
Initiation
Élongation
Termination
Initiation translation
1st step. Binding together an initiator tRNA carrying the amino acid methionine, the mRNA and small ribosomal subunit
Initiator tRNA has anticodon UAC
Small ribosomal subunit with its tRNA binds initially to the mRNA at the mRNAs 5’ end
Small subunit scans along the mRNA until it lines up over the start codon on the mRNA
Base pairing occurs between the tRNA anticodon and the start codon AUG on the mRNA. This pairing establishes the correct reading frame for translation of the codons along the rest of the mRNA. Essential as there isn’t “punctuation” along the mRNA that separates one codon from the next
Code is “commaless “
Large subunit binds onto small subunit, completing initiation stage of translation
Completed ribosomal has 3 sites that can accommodate tRNAs: E,P, and A sites
Ribosomal subunits consist of
Molecules of both ribosomal RNA ( rRNA ) and proteins
Although only a few rRNA are present in each ribosome, they make up about half the mass of the ribosome
Where are ribosomal subunits synthesized in eukaryotes
Nucleolus in nucleus
Leave the nucleus and become functional in cytoplasm
Methionine represents the
First of many amino acids joined together one at a time by the ribosome to form a long polypeptide chain
Translation key
E=exit site
P= peptidyl site
A= aminoacyl site
Elongation cycle adds
One new amino acid at a time
To the new forming polypeptide chain
Elongation
Translation
At the begging of each cycle. Polypeptide chain is held by the tRNA in the P site
At the end of the it’s held again by a new tRNA in the P site
Chain increases in length by one amino acid during each cycle
At the beginning of the first elongation cycle the P site is occupied by a charged tRNA with methionine attached to it. (E and A sites are empty)
A tRNA with a new amino acid enters the empty A site with an anticodon complementary to the codon exposed in the A site (charged tRNA is entering the empty A site)
Peptidyl transferase in the large subunit catalyzes the formation of a peptide bond between the 2 amino acids attached to the 2 tRNAs held in P and A sites
Ribosome now translocates along the mRNA a distance of 3 nucleotides in the 5’ to 3’ direction
This shifts the uncharged tRNA in the P site to the E site and the charged tRNA in the A site to the P site. A site left empty
Uncharged tRNA in the E site exits ribosome
Empty A site is no ready to receive a new tRNA bringing in the next amino acid to be added to the polypeptide chain
This ends one elongation cycle with the polypeptide chain again held by a new tRNA in the P site
Cycle is repeated over and over. Growing polypeptide chain 1 amino acid at a time.
The genetic code is non overlapping. This means successive triplet codons are read in order to not successive nucleotides
