ch 12
blast x take nucleotide sequence translate to protein sequence
methionine AUG start codon
amino acids are bonded by peptide bond
DNA Template strand- transcription to mRNA(made by DNA polymerase) to then go out of nucleus into translation in rRNA(ribosome) through tRNA(amino acids) and make protein
DNA coding strand- codes-codes for gene of interest
unambiguous- meaning that each codon specifies a single amino acid
codons is the mRNA template (the lock)
Anticodon is the tRNA strand (the one that brings the amino acids (key)
Degenerate: a codon can be thai which means that several triplet codons can be specific to a single amino acid. this is the case for 18 out of 20 amino acids
the code includes one start(initiate) and three stops (termination)
Francois Jacob and Jacques Monod postulated the existence of mRNA
the four ribonucleotides can specify 20 words (amino acids)
Sydney Brenner argued that the code had to be triplet since 3 letter word represent a minimal use of 4 letters to specify 20 amino acids 4^3=64
francis crick experimental evidence supported Sydney Brenner by using phage T4 they studied frameshift mutation
Marshall Nirenberg and J. Heinrich Matthaei deciphered the first specific coding sequences. They used in vitro (cell free in test tube) protein synthesizing system and an enzyme polynucleotide phosphorylase.
in cell free system- amino acids are incorporated into polypeptide chains
With RNA homopolymers- only one type of ribonucleotide, were added in vitro and deciphered UUU, AAA, CCC amino acids not GGG because it folds
RNA heteropolymers- adding more than one ribonucleotide
Triplet Binding Assay- developed by nirenberg and philip leader which led to assignments of triplets. triplet sequence acts like a codon in mRNA, attracting tRNA containing complementary sequence and carrying a specific amino acid
triplets, ribosomes bind to a single codon of 3 nucleotides
Illustration of the behavior of the components during the triplet binding assay. The synthetic UUU triplet RNA sequence acts as a codon, attracting the complementary AAA anticodon of the charged tRNA phe, which together are bound by the subunits of the ribosome
initiation codon is AUG encode methionine
Termination codons, UAA, UAG, UGA do not encode amino acids
Wobble theory: there is only 40 tRNA and 61 codons so that where wobble theory comes in place, meaning that the bases does not have to exactly match ex G with C. the third base is more relaxed and can bind to another base that does not match first position of the anticodon 5’ may pair with A or G at third position of mRNA codon 3’, and G may likewise pair with U or C
ordered genetic code: triplet patterns that share chemically similar amino acids share one or two middle bases: ex changing AAA to AGA both being different amino acids but both are hydrophobic
in bacteria AUG codes for methionine and is initiator codon?
nonsense mutation: when tRNA is translating to proteins if a mutation occurs that codes for a stop codon before the full polypeptide chain finishes and it ends earlier.
So, out of 64 codons only 61 encode for the 20 amino acids and 3 are stop codons
Walter Fries: confirm the degenerate, unambiguous, comaless, and start anf termination signals by using RNA containing bacteriophage MS2
coding is universal across eukaryots, porokaryots etc, except in mitochondria; DNA some codons don’t code for the normal amino acids, it varies a little bit
there are 22 tRNA encoded in human mitochondria
overlapping genes: when a single mRNA has several initiation points, so it creates several initiation frames and creating more than one protein. discover in X174
RNA polymerase: differnet from DNA polymerase in dna replication. this is an enzyme that catalyses rna from the dna template, the difference is that it contains ribose instead of dexiribose and it does not require a primer
NOTE: DNA replication and RNA transcription does not happen at the same spot at the same time, DNA happens in S phase while rna happens in Interphase outside S phase, and it happens in specific regions on the promoters, where RNA polymerase attaches.
NTP are substrates that catalyze polymerization of NMP and PPi
The core enzyme RNA polymerase consist of two betha polypeptides providing an active site for transcription while sigma is for initiation of transcription. sigma recognizes promoters to bind
two important promoters are -10 TATA prinbnow box and the other -35 TTGACA
After a few ribonucleotides are added, sigma dissociates from the holoenzyme and elongation continues under the core enzyme
Hairpin secondary structure, where the nucleotides fold back to itself causing a termination of the transcript.
Two types of hairpin- intrinsic termination- A weak pair with U to A leading to dissociation of RNA polymerase and the transcript is released. the other type is rho dependent factor which is a protein that dissociates RNA hairpins and DNA-RNA interactions. Rho breaks hydrogen bonds and causes dissociation of RNA polymerase and dna template and transcript separates.
Cistron: section in DNA that codes for one polypeptide in protein synthesis
Polycistronic mRNA is a mRNA that encodes the amino acid for several proteins, this is in prokaryotes
Differences between prokaryotic and eukaryotic transcription
In eukaryotes, transcription occurs in the nucleus and has to move out to get to ribosome
Transcription in Eukaryots occurs under 3 separate forms of RNA polymerase and bacteria is just one
RNA I and III transcribe tRNA and rRNA which are needed all the time for protein synthesis
RNA II, transcribes protein coding genes (mRNA) which is highly regulated
The following will be referring to cis acting in RNAP II
there are four cis elements. the first is Core Promoter- min. part of promoter includes 80 nucleotides including transcription start site. then we have proximal promoter element, located upstream helps modulate level of transcription. Last two are enhancers and silencers influencing efficiency of transcription rate. both of these can be located upstream, downstream, or internal. enhancers enhances transcription and silencer blocks, or reduces the transcription.
Cis acting element within core promoter is the Godberg-Hogness box also known as TATA box with 20-30 nucleotides, located -30 uspstream
In prokaryotes the TATA box is located -10 and binds directly to RNAP
Now talking about trans in RNAP II
known as transcription factors: there are two, general transcription factors (GTFs): required for transcription, load RNAP to DNA. Then there is transcriptional activators (enhancers) and repressors(silencers), influencing efficiency or rate of RRNAP II transcription initiation.
In the GTF, the so called TFIID binds directly to TATA box
eukaryotes require compact chromatin fiber and to unwind and uncompact the chromatids to make it accessible for transcription it is called chromatin remodeling. prokaryots do not have chromatids, they have round doble DNA called nucleoid
termination in bacteria is due to hairpin secondary structure and in eukaryots termination happens by polyadenylation.
polyadeylation cleaveage: involves CPSF and CstF factors to cut the RNA
it first starts by the C-terminal domain (CTD tail) on the RNA polymerase, the tail carries CPSF and CstF
then on the DNA gene, a sequence call Poly-A signal (AAUAAA) sequences which activates CPSF and CstF to adhere to RNA and cut it after 10 to 35 ribonucleotides.
Then, Poly A polymerase comes in into the 3’ OH end and ads a bunch of adines AAAAAA
Then poly-A binding protein attached to the poly A tail to prevent forn nucelases to degreating it
after it gets cut off the process continues and it is believe to stop because on the 5’ it does not have a cap(m7G) to terminate the process on the transcription,
in eukaryotes before mRNA is coded for protein it is a pre-mRNA that needs RNA splicing (adding cap and poly A tail ) to protect it from other enzymes
on the cap 7-methygluanosine is added to protect from nucelase attack
within the mRNA between the cap and tail there are introns and extrons, the extrons are coded and the introns are not. the Spliceosome (made of small nuclear RNA snRNA containing U1 U2…) binds to introns and cuts them out leaving only extrons and then mRNA is ready to be cut.
end of differences
RNA splicing
the first intron containing genes was B-goblin gene
the concept of alternative splicing- genes can encode for more than one protein , meaning proteins can encode more proteins than it has protein coding genes.
the process of cutting introns in tRNA involves intron being cut on both ends by a endonuclease and the exons glue together by a ligase
introns can be separated into groups
intron group I: happens in mRNA, tRNA, rRNA in bacteria and low euk and high plants. It is self excision, requires no help to cut, refer to ribozymes. the G factor guanosine (3’Oh) in the intron attacks the Phosphodiester bond (5’), then the OH and P of the extron join together
Intron II: aslo self excision in mRNA and tRNA in mitochondria and chloroplast in plants, fungi, bacteria
RNA EDITING
insertion/deletion editing: nucelotides added or deleted to or subtracted from total number of bases
Substitution editing: nucleotides are altered. common in mitochondrial and chloroplast RNA transcribed in plants
ex; of insertion / deletion is Trypanosoma, causing sleeping sickness caused by gRNA(guide RNA)