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Exam 3 Content, TATA-Binding Proteins (TBPs): recognize and bind to TATA…
Exam 3 Content
RNA Molecules and RNA Processing
Gene Organization
Colinearity:
proportionality of nucleotides to amino acids in encoded protein
Colinear:
number of nucleotides in a genes should be proportional to the number of amino acids in the encoded protein
bacteria and viruses
Noncolinear:
DNA much longer than RNA
eukaryotic genes
Exons:
coding DNA (will give rise to proteins)
Introns:
noncoding regions of DNA (located between exons)
categorized by how they are removed
Nuclear pre-mRNA: located in protein-encoding genes in the nucleus of eukaryotes
Structure of mRNA
Codon:
set of three nucleotides that specify an amino acid of a protein
Three Primary Regions
(both prokaryotic and eukaryotic mRNA)
5' Untranslated Region (5'-UTR):
a sequence of nucleotides at the 5' end of mRNA that does not encode any amino acids
in bacteria contains a consensus sequence called
Shine-Dalgarno sequence,
which serves as a ribosomal binding site
Protein-Coding Region:
compromises codons that specify the amino acid sequence of a protein
begins with start codon, ends with stop codon
3' Untranslated Region (3'-UTR):
a sequence of nucleotides at the 3' end of mRNA that does not encode any amino acids
affects mRNA stability and helps regulate the translation of mRNA protein-coding sequence
Pre-mRNA Processing (only in eukaryotes)
Pre-mRNA:
immature single strand of mRNA containing exons and introns
Modifications of pre-mRNA
1) Addition of 5' Cap:
a nucleotide with 7-methylguanine; 5'-5' bond is attached to the 5' end
Purpose
takes place rapidly after initiation of transcription
allows cap-binding proteins to attach to it so a ribosome can recognize and bind to it
increases stability of mRNA and influences removal of introns
Process
i) One of the three phosphate groups at 5' end of the mRNA is removed and a guanine nucleotide (with its phosphate group) is added
ii) Methyl groups are added to position 7 of the guanine base and the 2' position of the sugar in the second and third nucleotide
2) Addition of the Poly-A Tail:
50-250 adenine nucleotides added to the 3' end of the mRNA
Purpose/Process
not encoded in DNA, added in process after transcription in a process called
polyadenylation
endonuclease cleavage occurs 20 nucleotides downstream of AAUAA consensus sequence, then poly-A tail is added
increases stability of mRNA (can't leave nucleus without it)
facilitates ribosome binding
3) RNA Splicing:
removal of introns
requires 3 sequences in the intron
5' splice site
3' splice site
Branch Point:
an adenine nucleotide 18-40 nucleotides upstream of 3' splice site
takes place within
spliceosome
(large/complex molecular structure made up of 5 RNA molecules and 300 proteins)
small nuclear RNAs (snRNAs):
aid in the proccess
i) U1 attaches to 5' splice site
ii) U2 attaches to branch point
iii) U4, U5, and U6 join the spliceosome to form lariat
U1 and U4 dissociate
iv) 5 base pairing between mRNA and snRNA hold the spliceosome together
Process
i) mRNA is cut at 5' splice site
ii) 5' end of intron attaches to the branch point
iii) A cut is made at 3' splice site
iv) Intron is released as a lariat and the two exons are spliced together
v) The bond holding the lariat together is broken and it is degraded
vi) The spliced mRNA is exported to the cytoplasm and translated
Alternative Processing Pathways
Alternative Splicing:
the same pre-mRNA can be spliced in more than one way to yield different mRNAs that are translated into different proteins
either two introns are removed, or two introns and exon 2
Multiple 3' Cleavage Sites:
two or more potential sites for cleavage and polyadenylation are present in the pre-mRNA
may or may not produce a different protein depending on whether site is located before or after stop codon
can also change 3'-UTR which affects mRNA stability and translation
4) RNA Editing:
coding sequence of mRNA is altered after transcription so that the translated protein has an amino acid sequence that differs from that of the encoded gene
i) The pre-edited mRNA pairs with guide RNA
ii) The guide RNA serves as a template for addition, deletion, or alteration of bases
iii) Mature mRNA is released
Transfer RNA (tRNA):
attaches amino acids during protein synthesis
Anticodon:
sequence of 3 nucleotides complementary to mRNA
same as original DNA but U instead of T
Ribosomal RNA (rRNA):
component of the ribosome
Prokaryotic rRNA Processing
i) Methyl groups are added to specific bases and to the 2'-carbon atom of some ribose sugars
ii) The RNA is cleaved into several intermediates
iii) Intermediates are trimmed
iv) Results in mature rRNA molecules
All encoded by a single type of gene
Eukaryotic rRNA Processing
i) Methyl groups are added to specific bases and to the 2'-carbon atom of some ribose sugars
ii) The RNA is cleaved into several intermediates
iii) Results in mature rRNA molecules
Two types of rRNA genes: large and small
RNA Interference (RNAi):
powerful and precise mechanism used by eukaryotic cells to limit invasion of foreign genes and to censor the expression of their own genes
Small Interfering RNA (siRNA)
Origin: mRNA, transposon, or virus
Action: degredation of mRNA
Target: genes from which they were transcribes
MicroRNA (miRNA)
Origin: RNA transcribed from distinct gene
Action: inhibition of translation
Target: genes other than those from which they were transcribed
Transcription
RNA
evidence suggests RNA was the first genetic material
Ribozymes:
catalytic RNA
Thomas Cech, 1981
cut out parts of their own sequence
connect RNA molecules
replicate
catalyze formation of peptide bonds between amino acids (proteins)
RNA Structure
polymer of nucleotides (sugar, base, phosphate)
ribose on 2' carbon
A+U, G+C
Primary Structure
: single strand
Secondary Structure
folds (H-bonding)
stem/hairpin
variety of structures causes many different functions
Top 3 Classes of RNAs
Ribosomal RNA (rRNA)
Cell Type: bacterial and eukaryotic
Location: cytoplasm
Function: structural and functional components of ribosome
Messenger RNA (mRNA)
Cell Type: bacterial and eukaryotic
Location: nucleus and cytoplasm
Function: carries genetic code for proteins
Transfer RNA (tRNA)
Location: cytoplasm
Cell Type: bacterial and eukaryotic
Function: helps incorporate amino acids into polypeptide chain
Transcription is the synthesis of an RNA molecule from a DNA template
while replication copies the whole molecule, transcription only transcribes part of the DNA into RNA
Highly Selective:
individual genes transcribed only as needed
imposes fundamental problem on the cell: how to recognize individual genes and transcribe them at the proper place and time
3 Major Components
DNA Template
Raw Materials (ribonucleoside triphosphate)
provides energy to build new RNA molecule
Ribonucleoside Triphosphates (rNTPs):
substrate for transcription
each consist of ribose sugar and base (a nucleoside) attached to three phosphate groups
in RNA synthesis, added one at a time at 3' end (phosphodiester bond)
Transcription Apparatus
consists of proteins necessary to catalyze the synthesis of RNA
Template of gene undergoing transcription: Christmas tree model
DNase degrades trunk (DNA)
RNase degrades branches (RNA)
Template Strand:
nucleotide strand used for transcription (3'--->5')
Nontemplate Strand:
not transcribed (5'--->3')
Transcription Unit:
a stretch of DNA that encodes an RNA molecule and the sequences necessary for its transcription
Promoter:
DNA sequence that the transcription apparatus recognizes and binds to
determines which strand to use and the transcription start site (also the direction of transcription)
RNA-Coding Region:
sequences of DNA nucleotides that is copied into the RNA molecule
Terminator:
sequence that signals where transcription is to end
usually part of RNA-coding region
Bacterial Transcription
Bacterial RNA Polymerase:
catalyzes synthesis of all classes of bacterial RNA
Core enzyme
at the heart of bacterial RNA polymerases made up of five subunits
Alpha (two copies)
Beta (one copy)
Beta Prime (one copy)
Omega (one copy)
not essential for transcription
helps to stabilize the enzyme
Sigma subunit (not part of core enzyme)
controls binding of RNA polymerase to the promoter
binds to create holoenzyme
Holoenzyme binds to -35 and -10 consensus sequences in the promoter, creating a closed complex
Consensus sequences compromises the most commonly encountered nucleotides at each site
"N" means none in particular are most common
Mutations at consensus sequences would reduce transcription activity
Without sigma subunit RNA polymerase would initiate transcription at a random point
Steps of Bacterial Transcription
1. Initiation:
when the transcription apparatus assembles on the promoter and begins RNA synthesis
i) Promoter Recognition
Bacterial Promoter:
sequences recognized by the transcription apparatus (holoenzyme)
required for transcription
tells where to start, what strand to use, and direction of transcription
ii) Formation of the Transcription Bubble
Holoenzyme binds to promoter and unwinds the double-stranded DNA
iii) Creation of the First Bonds Between rNTPs
a nucleotide triphosphate (rNTP) complementary to the DNA serves as the first nucleotide in the RNA molecule
does not require a promoter
iv) Escape of the Transcription Apparatus from the Promoter
2. Elongation:
carried out by the action of RNA polymerase
3. Termination:
stop synthesizing, RNA released from RNA polymerase, RNA separated from DNA, RNA polymerase detaches from DNA
Rho-Dependent Termination:
uses rho factor
i) Rho binds to rut site and moves toward 3' end
Rut site: rho utilization site
ii) When RNA polymerase encounters a terminator sequence it pauses and rho catches up
iii) Using helicase activity, rho unwinds the DNA-RNA hybrid and brings an end to transcription
Rho-Independent Termination:
hairpin structure formed by inverted repeats, followed by a string of uracil
i) A rho-independent terminator contains an inverted repeat followed by a string of approximately 6 adenine nucleotides
ii) The inverted repeats are transcribed into RNA
iii)The string of Us causes the RNA polymerase to pause
iv) The inverted repeats in RNA fold into a hairpin loop, which destabilized DNA-RNA pairing
v) The RNA transcript separates from the template, termination transcription
Eukaryotic Transcription
Differences Between Bacterial and Eukaryotic Transcription
Polymerase(s)
Bacterial: one polymerase
Eukaryotic: 3 polymerases
Promoter(s)
Bacterial: generic promoter
Eukaryotic: promoter depends on polymerase
Promoter Recognition/Initiation
Bacterial: recognized by holoenzyme (part of bacterial polymerase)
Eukaryotic: many accessory proteins take part in the binding of eukaryotic RNA polymerases
different types of promoters require different proteins
Nucleosomes
In eukaryotes, DNA is complexed with histones that make it inaccessible for transcription
Chromatin structure must be modified prior to transcription by several proteins in order for it to take place
Eukaryotic RNA Polymerases
RNA Polymerase I:
transcribes large rRNAs
RNA Polymerase II:
transcribes pre-mRNA
also snoRNAs + some snRNAs and miRNAs
RNA Polymerase III:
transcribes tRNAs and small rRNAs
also some snRNAs and miRNAs
Promoters
Basal Transcription Apparatus:
combination of general transcription factors with RNA polymerase
General Transcription Factors: a type of accessory protein (subcategory of "transcription factors" specifically for the formation of basal apparatus
Assembles near transcription start site and is sufficient to initiate minimal levels of transcription
Types of Promoters Recognized by RNA Polymerase II
Core Promoter
Located immediately upstream of the gene
Includes one or more consensus sequences
TATA Box:
most common of these consensus sequences
Sequence TATAAA
25-30bp upstream of start site
Regulatory Promoter
Located immediately upstream of core promoter
A variety of different consensus sequences can be found in regulatory promoters, and they can be mixed and matched in different combinations
Transcription factors bind to these sequences and, either directly or indirectly, make contact with the basal transcription apparatus and affect the rate at which transcription is initiated.
RNA Polymerase I and III have their own unique promoters
Steps of Eukaryotic Transcription
1. Initiation:
intiated through assembly of transcription machinery on the promoter
Machinery consists of RNA polymerase II and a series of transcription factors
i)
Begins when regulatory proteins bind DNA near the promoter and modify chromatin structure
ii)
Transcription factors and RNA polymerase II bind to the core promoter
iii)
Transcription activator proteins bind to sequences in enhancers
enhancers initiate higher levels of transcription after basal apparatus
iv)
DNA loops out, allowing the proteins bound to the enhancer to interact with basal transcription apparatus
v)
Transcriptional activator proteins bind to the sequences in the regulatory promoter and interact with basal apparatus through mediator
recruits and stabilizes the basal machinery to the promoter
2. Elongation
i)
RNA polymerase maintains a transcription bubble
ii)
DNA double helix enters a cleft in the polymerase and is gripped by the enzyme
iii)
The two strands of DNA are unwound and complementary nucleotides are added to the growing RNA molecule
iv)
As it funnels through the polymerase, the DNA-RNA hybrid hits a wall of amino acids and bends at almost a right angle
this bends the position of the DNA-RNA hybrid at the active site of the polymerase, where new nucleotides are added to the 3' end of the RNA
v)
The newly synthesized RNA is separated from the DNA and runs through another cleft in the enzyme before exiting the polymerase
many transcription factors are left behind and can serve to quickly reinitiate transcription with another enzyme
3. Termination:
different mechanisms for each RNA polymerase
RNA Polymerase I: requires termination factor like rho to bind to DNA sequence
RNA Polymerase II: termination does not occur at specific sequences, instead RNA pol II often continues to synthesize further than needed
Rat1 then works backward, degrading the extra nucleotides, until it reaches promoter, where transcription is terminated
RNA Polymerase III: ends transcription after a sequence of Us is transcribed (like rho-independent)
Control of Gene Expression in Bacteria
Genes and Regulatory Elements
Structural Genes:
encode proteins used in metabolism, biosynthesis, and structural roles
Regulatory Genes:
produce RNA or proteins that interact with other DNA sequences and affect the transcription or translation of those sequences
Positive Control:
stimulates gene expression (on)
Negative Control:
inhibits gene expression (off)
DNA Binding Proteins
Domains:
discrete functional parts of regulatory proteins responsible for binding to DNA
Motif:
simple structure within DNA binding domain that fits into the major groove of the DNA
Inducible:
transcription normally off
Repressible:
transcription normally on
Types of Operons
Negative Inducible
transcription off due to repressor at operator site
must be turned on by inducer
Negative Repressible
transcription on due to inactive repressor at operator site
must be turned off by corepressor
Positive Inducible
transcription off due to inactive activator
must be turned on by inducer
Positive Repressible
transcription on because activator binds readily to DNA
must be turned off by some substance that deactivates activator
Lac Operon
Negative Inducible
Inducer: allolactose
Regulator Gene (lacI): encodes repressor
Absence of Lactose
regulator protein inhibits transcription
Presence of Lactose
allolactose deactivates repressor, allowing from transcription
Lac Mutations
Regulatory Gene (LacI) mutations are trans acting (affect other genes)
Operator Gene (LacO) mutations are cis acting
Promoter Mutations (LacP) are cis acting
partial diploid (F plasmid and chromosome are different)
Structural Gene Mutations (lacZ/lacY) only affect themselves
Trp Operon
Attenuation:
termination of transcription early before it reaches structural genes
Attenuator:
terminates transcription
Antiterminator:
prevents termination
Levels of Tryptophan
High Levels
Ribosome does not stall at Trp codon
Ribosome covers region 2 when region 3 is transcribed
Secondary Structure of 5'-UTR: 3+4 hairpin
Terminates transcription of trp operon
Low Levels
Ribosome stalls at Trp codons
Ribosome covers region 1 when region 3 is transcribed
Secondary Structure of 5' UTR: 2+3 hairpin
Does NOT terminate transcription of trp operon
Genetic Code and Translation
Proteins:
Polymers composed of amino acids liked by peptide bonds
Peptide Bonds:
formed between carboxyl and amino ends of amino acids
Primary Structure:
amino acid chain
Secondary Structure:
folding of amino acid chain
Tertiary Structure:
further folding
Quaternary Structure:
two or more polypeptide chains associate
Degeneracy of Code
Degenerate Code:
the code contains more information that is needed to specify the amino acid (amino acid may be specified by more than one codon)
Sense Codons (61):
encode amino acids
Amino Acids (20)
Stop Codons (3);
UAA, UAG, UGA
Isoaccepting tRNAs:
different tRNAs that accept an amino acid despite having different anticodons
Occurs due to
Wobble Hypothesis
(third base in a codon binds weakly and is flexible
Ex. G in the anticodon can pair with C or U in the codon
Only with G and U in anticodon
Translation:
protein synthesis
1) tRNA Charging:
binding of tRNA molecules to their appropriate amino acids
i) Amino acid reacts with ATP, producing aminoacyl-AMP and PPi
ii) Amino acid is transferred to tRNA, where the CCA sequence of tRNA binds at the adenine to the carboxyl group of the amino acid
iii) AMP is released
Aminoacyl-tRNA Synthetases:
a set of enzymes that are the key to specificity between amino acids and their tRNAs
a cell has 20 aminoacyl-tRNA synthetases, ones for each amino acid
2) Initiation of Translation:
assembly of mRNA, small and large subunits of ribosome, initiation factors, initiator tRNA, and GTP
Bacteria
i) IF-3 binds to the small subunit, preventing the large subunit from binding
this allows the small subunit to attach to the mRNA
Shine-Dalgarno
sequence is required for attachment of small subunit
ii) a tRNA charged with N-formylmethionine forms a complex with IF-2 and GTP (IF-2 brings GTP)
binds to initiation codon while IF-1 joins the small subunit
iii) All initiation factors dissociate from the complex and GTP is hydrolyzed to GDP
iv) The large subunit joins to create 70s initiation complex
Eukaryotes
i) Kozak sequence facilitates identification of the start codon
ii) Proteins that attack to the poly-A tail interact with cap-binding proteins
this enhances the binding of the ribosome to the 5' end of the mRNA
3) Elongation:
Attachment of amino acids to form polypeptides
i) fMET-tRNA occupies the P site of the ribosome
ii) EF-Tu, GTP, and charged tRNA form a complex that enters the A site of the ribosome
iii) GTP is cleaved to GDP and the EF-Tu-GDP complex is released, leaving the charged tRNA in the A site
EF-Ts regenerates the EF-Tu-GTP complex, which is then ready to combine with another charged tRNA
iv) A peptide bond forms between the amino acids in the P and A sites, and the tRNA in the P site releases its amino acid
v) The ribosome moves down the mRNA to the next codon (translocation) which requires EF-G and GTP
vi) The tRNA that was in the P site is now in the E site from which it moves into the cytoplasm
vii) The tRNA that occupied the A site is now in the P site, leaving the A site open to receive another tRNA
4) Termination:
protein synthesis ends when ribosome translocates to a terminal codon
i) When the ribosome translocates to a stop codon, there is no tRNA with an anticodon that can pain with the codon in the A site
ii) Release Factor (RF) 1 or 2 depending on the codon, attaches to the A site
iii) RF3 forms a complex with GTP and binds to the ribosome
iv) The polypeptide is released from the tRNA in the P site
v) GTP associated with RF3 is hydrolyzed to GDP
vi) The tRNA, mRNA, and release factors are released from the ribosome
TATA-Binding Proteins (TBPs)
: recognize and bind to TATA Box