Lecture 8 Flashcards
RNA can be used to
1) translated into proteins
2) RNA is the endpoint,
i.e. “non-coding” RNAs, ncRNA’s ((e.g. tRNA, rRNA, microRNA’s
regulatory function
ncRNA’s are often ___ achieve their final function
modified and combined with proteins to
(e.g. ribosomes are rRNAs bound with many proteins).
However, RNA is more ___ then DNA
structurally diverse and can take on a myriad of shapes (ex. classic stem-loop)
RNA vs DNA (differences)
1) RNA sugar molecule retains its oxygen (this just ribose)
2) uracil replaces thymine (lacks a methyl group) can be bound to both adenine and guanine
3) RNA is initially single stranded
Nucleosides vs nucleotides
Nucleosides - RNA base along with the sugar
Nucleotides - nucleosides with the triphosphate.
Transcribed sections of DNA (aka ____) can be on either strand of the DNA.
but RNA synthesis is always from
aka genes
DNA’s 3’ to 5’. Each new RNA base attaches on the 3’ end of the RNA.
RNA bases are added by
the removal of H on the 3’ hydroxyl of the proceeding ribose sugar (S), and bonded to the alpha phosphate (P) on the next ribose.
phosphodiester bonds are covalent
rxns are powered by the two phosphates that are lost in the process
famous electron microscopy photo of transcription (1970) from ___
This was a critical piece of evidence that showed ____
Miller, Hamkalo, and Thomas
showed RNA transcriptions coming off of a central DNA molecule
transcription can be ___ meaning a second transcript can begin before the first has finished
internally continual
Transcription Initiation & Termination site
branches are longer in one direction (closer to the termination site)
Transcription in Prokaryotes and Eukaryotes are thematically very similar because
1) A specific promoter region is recognized and bound to by transcription factor or factors
2) Transcription factor(s) helps to recruit and position the much larger transcription apparatus that includes the RNA polymerase
3) The polymerase complex complements the template strand by adding bases on the 3’ end of the transcript
4) Transcription is terminated when the termination site is reached
Eukaryotes have extensive modification of mRNA including ____
Bacteria have ____
poly-A tail, methyl guanine capping, splicing, and editing.
Bacteria have very little post transcriptional modification, but a very few do have a short poly-A tail.
Bacteria can have multiple genes (proteins) contained in a single transcriptional unit termed an ____
is termed _____
operon - An mRNA of multiple genes
poly cistronic mRNA.
Bacterial mRNA translation is often begun before transcription is completed. These processes are ____ in Eukaryote
physically separated
Bacteria vs Eukaryotic mRNA polymerase
Bacteria - one
Eukaryotes - three that specialize
Eukaryotic RNA Polymerases
Polymerase IV and V are only seen in plants.
1 Large rRNAs
2 all pre RNAs, snoRNAs (some mi and sn RNAs)
3 Small RNAs, t RNAs (some mi and sn RNAs)
Promotor sequences in Prokaryotes and Eukaryotes
Prokaryotes - tend to be immediately adjacent to the coding sequence
Eukaryotes - can be more distant
Transcription is initiated at specific locations by
recognition of particular promotor sequences
In Prokaryotes, two common promotor sequences are
TTGACAT at the -35 region and TATAAT at the -10 region.
actual promotion sequences vary, thus you can think of the TATAAT as a “average” also termed a consensus sequence.
The promotor regions are not transcribed in mRNA.
consensus sequence
(Prokaryotes)
TATAAT as a “average” also termed a consensus sequence
Prokaryote Initiation
a multi-protein complex, the RNA polymerase holoenzyme, scans the DNA for promotor sequences
the Sigma70 protein is critical to identify the promoter and initial binding to the -35 and -10 regions
Binding to the promoter sequence positions the complex so it begins transcription at the proper base.
sigma factors in bacteria
are many that recognize different promoter sequences
but the holoenzyme is the same
Prokyarotic transcription often “false starts” a few times producing
2-6 nucleotides while still bound to the promoter then backing up to the +1 position. (those few RNA nucleotides are released.)
After 9 to 12 nucleotides are transcribed the sigma factor is released, this aids in the complex no longer being able to bind to the promotor.
Prokaryote transcription “bubble” is
relatively small, only about 18 bases of unwound DNA
Prokaryote Elongation
about 8 to 10 RNA’s are bound to the DNA at any one time before they are pulled off
continues at a rate of ~ 40 bases a second, and the same locus can be translated more than once (sequentially) at the same time
can pause (or stall and the complex disassociate) for a number of reasons.
Prokaryote Elongation can pause because of
One group of these events is termed “backtracking”, this is a normal but unintentional act by the RNA polymerase.
This does highlight that the RNA polymerase can slide forward and backward a bit in its normal action.
cells generally try to prevent backtracking (because may cause addition of incorrect basses)
RNA polymerase can back up and replace a mismatched RNA base
Prokaryote Termination should be though of as more a ____
slowing process than a sudden stop
Two modes for prokaryote termination
1) Rho – Independent
2) Rho – Dependent
Rho – Independent termination
A particular sequence forms a loop called a hairpin before a RNA-DNA hybrid weak area (ex. many A-Us)
Because RNA polymerase to back up if the RNA-DNA hybrid is weak (ex. when there are many A-U’s) the RNA polymerase backs up into the hairpin
Backing up into the hairpin causes the polymerase to detach
Rho – Dependent termination
the protein Rho winds around the RNA transcript
Rho eventually pulls the RNA transcript from the DNA
causes the polymerase to disassociate from the strands and terminate elongation.
to avoid stalling transcription in eukaryotes
proteins are modified and/or slid down the chromosome (chromatin remodeling)
degree of chromatin remodeling is one way that transcription and thus protein synthesis is up and down regulated.
transcription in Eukaryotes takes place in
the nucleus
translation is physically separated and occurs outside of the nucleus
factors that come together to influence pre-mRNA synthesis. These factors can bind to ___and ___
(Eukaryote)
(there can be 50 or more factors)
particular sites both immediately adjacent to the DNA site
and other sites much more distance (both upstream and downstream)
Eukaryotic Initiation
Polymerase II action begins with TFIID (transcription factor IID) (is essential) binding to the TATA box area (in the core promoter) (Promotor Sequences - can be more distant)
TFIID consists of numerous polypeptides, one of which is TBP
binding of the TFIID complex causes a change in the shape of the DNA, partially unwinding the DNA.
Once TFIID is bound to the TATA box, it helps to recruit many other transcription factors (TF), including
o RNA Polymerase II
o Transcriptional “mediator”
TBP
TBP (TATA binding protein) is the part that recognizes and binds (in the minor grove in the DNA) to the TATA sequence.
Transcriptional “mediator”
a large multi-poly (30+) peptide complex that regulates transcription through by binding to a distant transcription factor (that is bound to an “enhancer” region)
important for regulation
in Eukaryotic Initiation, After successful initiation (non-false starts) ____
This helps prevent ____
a number of proteins disassociate from the complex
prevent the complex from rebinding to the promotor region
some proteins (e.g. TFIID) that were critical to initiation remain at the initiation site and are able to induce another round of transcription
Eukaryotic Elongation
is enhanced by positive transcription elongation factor (P-TEFb) binding to the complex.
P-TEFb phosphorylates Polymerase II as well as DSIF and NELF (negative elongation factor).
CTD (carboxyl terminal domain) - helps regulate RNA processing including the capping of the pre-mRNA.
Without this phosphorylation by ____ DISF and NELF would _____
P-TEFb
would inhibit elongation
Eukaryotic Termination
RNA polymerase II - keeps adding RNA bases beyond the coding sequence
(the pre-mRNA is cleaved while this happens)
Rat1 then begins exonuclease activity that will eventually cause the polymerase to dissociate from the DNA
Eukaryotes modification of mRNA
is extensive
including poly-A tail, methyl guanine capping, splicing, and editing
Eukaryotic RNA polymerase I
specializes in producing the large ribosomal subunits
Ribosomal genes in these regions of the DNA are highly repetitive allowing for rapid increases of ribosomes in preparation for cell division.
The factors differ from those used in polymerase II
Eukaryotic RNA Polymerase III
transcribes non-coding RNAs (ncRNA) shorter than 300bp (including the small rRNA’s)
there are three types of promoters:
o type 1: produces 5S rRNAs
o type 2: tRNAs,
o type 3: small nuclear RNA (involved in pre-mRNA processing)
RNA Pol 1 termination
terminated by particular protein factors –> bound directly to DNA
RNA Pol III termination
terminates after a long series of U’s (poly U)
