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147 Cards in this Set

  • Front
  • Back
what are the structures of a nucleotide
-purine or pyrimidine base
-pentose (5 carbon ring)
-phosphate grp
what are the purine Heterocyclic bases? (2)
-adenine
-guanine
what are the pyrimidine Heterocyclic bases? (3)
-cytosine
-thymine (DNA)
-uracil (RNA)
the two conformations of the ribose are? and when are they found in equilibrium? and when are only the cyclic forms found?
-5 carbon aldehyde
-cyclic beta-furanose
-in solution
-in DNA and RNA
how many conformations of ribofuranose (and deoxyribofuranose) are there?
-4 total
-2 for ribofuranose at the 2C
-2 for deoxyribofuranose at the 3C
what is the suffix for nucleotides?
what is the suffix for nucleosides?
(for purines)
-ylate e.g. adenylate
-side e.g. adenoside
what is the suffice for nucleotides?
what is the suffix for nucleosides?
(for pyrimidines)
-ylate e.g. uridylate
-idine e.g. uridine
DNA is a polymer of 4 deoxynucleotides what are they
(hint A T G C)
-Deoxyadenosine
-Deoxyguanosine
-Deoxythymidine
-Deoxycytidine
Deoxyribonucleotides (and ribonucleotides) are linked via a what?
-a phosphodiester linkage
DNA is composed of two ______ strands paired and determined via unique _______ _______ patterns
-deoxynucleotide
-hydrogen bonding
DNA strands run parallel or anti-parallel?
- anti-parallel
-5' -> 3' run in opp. directions
Most DNA are ____ handed. alpha or beta form? most or least stable?
-right
-beta
-most
DNA is in the form of a _____ helix
double
the base pairs are stacked horizontally in the structure revealing _____ and _____ grooves
major and minor (broad and narrow)
what are the 3 forms of DNA?
of these three which two are right handed and which is left handed?
A, B and Z
A and B are right handed
Z is left handed
two unusual sequence structures that occur within DNA?
palindrome
mirror repeat
single strand palindromes create what?
double strand palindromes create what?
hairpins
cruciforms
RNA differs from DNA is 4 ways
-the sugar is ribose (hydroxyl grp at the 2 position)
-RNA has base U instead of T. U is exactly same as T except lacks methyl grp at pos. 5
-RNA is usually single stranded
-RNA is less stable than DNA
What are the multiple forms of RNA?
-tRNA
-catalytic RNA (ribozyme)
-regulatory RNA (miRNA)
-protein coding RNA (mRNA)
What are the 4 ribonucleotides of RNA?
-Adenosine
-Guanosine
-Uridine
-Cytidine
DNA denaturation (melting) measures what? the stability is determined by (4)?
the stability of double stranded polynucleotides
1. soln conditions
2. base pairing
3. base stacking
4. charge shielding
the term used to define - strand seperation increases UV absorption
hyperchromicity
T or F denaturation on large DNA strands is local?
true
3 correlations of denaturation
-denaturation % increases with temp
-the higher the base comp. the higher the Tm
-the longer the chain length the more stable
DNA renaturation (annealing/hybridization) exp. examines whether two single strands can from complimentary base pairs to give a stable double stranded structure. Base pairing is involved in (4)?
-gene expression and recomb
-brings molecules together
-key req for some activities (DNA polymerase)
-is used to id. sequences
nucleic acid annealing occurs optimally
25 degrees below the Tm
what is a good way to visualize DNA supercoiling?
a corded telephone where it usually is one coil, but when gets twisted wraps around itself
superhelical DNAs are?
circular DNAs with entirely intact strands (no breaks in the phosphodiester bonds; these are called closed circular DNAs) have their strands topologically linked (they can not be pulled apart without breaking one or more phosphodiester bonds); and the number of links (linking number, L) is fixed. If the linking number (L) is changed, the resulting strain is compensated for by introducing superhelical twists or writhe (W). This phenomenon is mathematically expressed: L = T + W.
definition of closed circular DNAs?
no breaks in any of the phosophdiester bonds
defintion of topologically linked?
they cannot be pulled apart without breaking one or more phosphodiester bonds
what do the letters in the equation L = T + W stand for?
L = linking number = number of links
T = Twists
W = Writhes
supercoiling writhe or twist
writhe = looks like a spring
twist = looks like party ribbon decorations twisted
what is a topoisomerase?
enzyme that controls superhelicity.

also catalyze phosphoryltransfer reactions
two types topoisomerases?
Type 1 - nicks the DNA and relaxes negative supercoils one turn at a time
Type 2 - also know as gyrase, in presence of ATP hydrolysis introduces negative supercoils, this enzyme makes transient double stranded breaks
chromatins?
basic proteins (eg histones) bind to DNA and form supramolecular nucleoprotein structures
nucleosomes are like ____ on a string
beads
Enormous length of human DNA
each diploid cell contains 2 meters of DNA
each human contains 2x10^11 KM of DNA
end to end 600 round trips to sun
chromosomes are very _____ molecules often _____ in shape
large
circular
what is the sole purpose of the genome-template?
for the transfer of information
problems related to the large size of chromosomes?
topological/mechanical problems
DNA replication is?
is a semi conservative process in which information transfer occurs through base pairing
DNA replication is ________
bidirectional
DNA polymerase; substrate? template? primer?
substrate = dNTPs
template = DNA strand that provides base pair information
primer = RNA or DNA base paired to template with a free 3’OH group. The 3’OH provides the nucleophile for the phosphoryl transfer reaction (new nucleotides are only added to the 3’ end) giving polarized DNA synthesis (5’ to 3’ direction only)
DNA polymerase uses base pairing and phosphoryl transfer reaction. the phosphoryl transfer reaction occurs as follows R, R" and R' what are the R's
R = primer
R" = the nucleoside that is being added
R' = the pyrophosphate that is released
DNA polymerase facilitates nucleophilic _______ and base _______
activation
insertion
DNA polymerase has several enzymatic activities T or F
true
5' to 3' exonuclease is?
Associated with DNA polymerase is a 5’ to 3’ exonuclease activity. A nuclease cleaves phosphodiester bonds via a theme 4 (phosphoryl transfer) mechanism (see below). An exonuclease requires and works from an RNA or DNA primer strand. A 5’ to 3’ exonuclease works from a 5’ end and proceeds toward the 3’ direction.
the initiation of DNA replication involves the synthesis of _______. DNA replication initiation occurs at a very specific site called ____.
primers
ori
completing lagging strand synthesis is ____ steps. and requires what two enzymes?
two
DNA polymerase 1 and DNA ligase
DNA ligase is used for what?
DNA ligase is a key enzyme that completes lagging strand synthesis.
It functions to link the short lagging strand products together into an intact strand. DNA ligases also function via the phosphoryl transfer reaction
Point mutations
Point mutations are substitutions of one base pair for a second. Transitions (A/T to G/C and G/C to A/T) maintain the same purine/pyrimidine orientations while transversions (A/T to T/A, G/C to C/G, A/T to C/G, C/G to A/T) reverse the purine/pyrimidine orientation.
small scale deletions or or insertions
Small scale deletions or insertions. If these small scale changes add or subtract one or more base pairs (but not a multiple of three) they are frequently called “frame shift” mutations (discussed in lecture 9).
large scale mutations
Large scale mutations include deletions, inversions, insertions and chromosome fusions
two causes of small scale mutations
- incorrect base incorporation during DNA replication
- chemical changes of bases in DNA molecule

(This typically involves the action of a mutagen (often a carcinogen). Examples include: the DEAMINATION of cytosine by nitrous acid (C gives U which base pairs like T), ALKYLATION of bases such a G by agents such as dimethylsulfate (to give O6-methylguanine that acts like A), DEPURINATION (removal of purine bases) by hydroxyl radicals, FORMATION of thymine dimers resulting from UV radiation.)
some chemical mutagens
NaNO2 (sodium nitrite) NaNO3 (sodium nitrate) and nitrosaime are nitrous acid precursors
deamination
loss of N grp, replaced with O and H put on Ring N
DNA Damage Repairing-proofreading
A 3’ to 5’ exonuclease activity is associated with DNA polymerases. The 3’ to 5’ exonuclease selectively removes nucleotides that have just been incorporated if they can not base pair with the template. Thus it removes mis-incorporated bases. The 3’ to 5’ exonuclease functions through a theme 1 (base pairing) and theme 4 (phosphoryl transfer) mechanisms.
the mismatch repair system
Repair systems exist that recognize mismatches or damaged bases, remove a stretch of newly synthesized DNA containing the damage, and repair the gap by DNA polymerization and ligation.
Direct repair demethylation
removing of the methyl from the protein
consequence of alkylation mutagenesis
incorrectly paired thymidylate nucleotide
Direct repair - fixing Thymine dimers
enzymatic splitting of thymine dimers and dealkylation of bases
recombination repair
in cases where the replicating fork is stalled by DNA dmg. recombination can repair the DNA. recombination repair involves theme 1 and 4
RecA
is a protein essential for facilitating the strand exchange involved in recombination. in homologous recombination
The Holliday struct.
The Holliday structure is an intermediate in homologous recombination in which the duplexes are joined by an “X” cross over. The “X” can migrate transferring strands between duplexes. The migration involves forming and braking base pairs (theme 1), and resolving a Holliday structure involves breaking phosphodiester bonds (theme 4).
homologous genetic recombination in eukaryotes
using homologous recombination during meiosis to exchange genetic material
resolution of holliday structures (cleavage)
horizontal cleavage = nonrecombinant ends
verical cleavage = recombinant ends
Programmed large scale changes
an important process in evolution, genetic diseases, such as cancer and AIDS and in gene formation. also necessary for normal processes
site specific recominbation
the bacteriophage lambda example

There are also normal cellular functions that involve site specific recombination to change the chromosome structure. An example is the antigenic phase variation that some bacteria display.
what is the bacteriophage lambda example and what is it an example of?
After bacteriophage (bacterial virus)  infection, there are two possible outcomes; either a lytic response (the virus multiplies and kills the bacterium) or the lysogenic response (the virus genome forms a stable association with the host genome). A critical step in the lysogenic response is the integration of the viral DNA into a precise location on the bacterial chromosome. This integration involves a recombination event between a specific location on the circular viral chromosome and a very precise site on the bacterial chromosome. The two sites, called attachment (att) sites, have 15 base pairs homology. The integration event is catalyzed by a viral protein (INT). The viral chromosome can also excise precisely (catalyzed by INT and XIS).

it is a site specific recombination
DNA transposable elements
DNA transpositition
retroviruses
retrotransposons
Transposition involves the following steps (6)?
a) Transposase binds to the short specific end DNA sequences (theme 2).
b) End bound transposase oligomerizes to form a “synaptic” nucleoprotein complex (theme 3).
c) DNA strands adjacent to bound transposase are cleaved in a 3 step process (OH from water, the 3’OH from cut DNA and OH from water act as nucleophiles in the phosphoryl transfer reactions (theme 4)).
d) The Transposase-DNA complex binds to target DNA (sequence specificity?).
e) 3’OH groups of the transposase bound transposon DNA performs a phosphoryl transfer (theme 4) attack on target DNA thus integrating the DNA.
f) DNA polymerase and ligase seal gaps (themes 1 and 4).
two pathways for transposition are?
direct and replicative
Generating antibody diversity
The capacity to develop cell lineages that can produce antibodies against a huge variety of antigens is due to the immune system’s progenitor cells undergoing specific types of genome changes that generates a huge variety of antibody encoding genes. These changes include chromosome rearrangements (mechanistically very similar to DNA transposition) that assemble genes from 3 gene segments.
Themes 2, 3 and 4 are involved in this process.
A high level of point mutagenesis also occurs.
recombination repair
in cases where the replicating fork is stalled by DNA dmg, recombination can repair the DNA. involves theme 1 and 4
transcription
is the process by which RNA is synthesized using a DNA template. RNA polymerase is the enzyme that performs transcription

nNTP (ATP, CTP, GTP & UTP) goes to RNAn residues + (n-1)PPI

All four themes are involved in this process.
RNA polymerase
RNA synthesizing enzyme. incorporates ribonucleotides in response to DNA template. it is also capable of initiating RNA synthesis de novo. . The DNA sequences required for transcription initiation are called promoters.
Sigma 70 holoenzyme
The E. coli  70 holoenzyme is the best studied RNA polymerase and serves as a model for understanding the properties of other RNA polymerases. The holoenzyme is composed of the core enzyme and an additional subunit called sigma (). The core enzyme is composed of four subunits (2 alpha (), 1 beta (), and 1 beta’ (’)). The core enzyme can catalyze the elongation reaction (the active site is shared by  and ’) but it can not initiate RNA synthesis properly. The  subunit is responsible for providing initiation specificity; the majority of promoter contacts reside in the  subunit. The  70 holoenzyme is the major form of RNA polymerase found in E. coli.
alternative sigma factors
Other sigma factors exist in E. coli. Each type of sigma factor gives the resulting holoenzyme a unique transcription initiation specificity. These alternative holoenzymes are less abundant than the  70 holoenzyme and are responsible for synthesizing special classes of mRNAs
other organisms (RNA polymerase)
have different RNA polymerases. Eukaryotic cells have 3 forms of RNA polymerase; some viruses such as T7 encode specialized RNA polymerases. Each type of RNA polymerase has its own initiation specificity (recognizes different DNA sequences as promoters).
The Sequence of the RNA Transcript Recapitulates the NonTemplate ____ Strand
DNA
Promoters
are DNA sequences that are recognized by RNA polymerase during transcription initiation. These sequences have been identified by: genetics, biochemistry, sequence comparisons, protection
Transcription initiation
-Genetics
-Biochemistry
-Sequence comparisons
-Protection
Genetics – the isolation of cis-active mutations that change the frequency of transcription initiation.
Biochemistry – the identification of DNA fragments that will program transcription initiation by RNA polymerase.
Sequence comparisons – common DNA sequences are found near the RNA start points (equivalent to the RNA 5’ end).
Protection – what DNA sequences does RNA polymerase “cover” when bound, ready to start?
What are the four steps to transcription initiation?
Closed complex formation. RNA polymerase binds to the double stranded DNA promoter. (R + P goes to RPc)

Open complex formation. The RPc undergoes a series of isomerization steps to form an open complex in which there is localized denaturation of the DNA near the start site. The template strand of the DNA is located in the active site of the enzyme. (RPc goes to RPo).

Initiation. Ribonucleoside triphosphates are condensed to form short oligonucleotides complementary to the template strand. (RPo + rNTPs yields short oligonucleotides + RPI)

Promoter clearance. The sigma subunit and the promoter are released, and core RNA polymerase starts extending the oligonucleotide to a long RNA molecule. (holoRPI–oligonucleotide + rNTPs yields core RPe-RNA + sigma)
Themes Associated with Transcriptional Initiation
Transcription initiation involves a theme 2 event (RNA polymerase binding to the promoter) to form a theme 3 nucleo-protein complex that leads to theme 4 catalysis (RNA synthesis) that requires theme 1 base pairing.
transcription occurs on ____ strands
both
two types of transcription termination?
Self (rho-independent) termination - DNA sequences that encode in the RNA a G-C rich hairpin structure followed by a run of UUUUU, destabilize the RDe-RNA complex leading to termination

Protein stimulated termination - A bacterial protein (p or rho) is known to stimulate transcription termination at other specific regions. p probably binds to certain types of RNA sequences just after they are made and then interacts with the RNA polymerase with which they are associated.
Eukaryotic transcriptional initiation requires _____ protein components
multiple
Initiation Factors ________ Associate and then Dissociate From the Promoter
temporally
Adaptive enzyme synthesis, what are the two types of regulation?
Two types of adaptive enzyme synthesis gene regulation exist. Induction is the turning on of gene expression in response to the presence of a compound related to the substrate of the encoded enzyme. The lactose (lac) operon is an example of an inducible system. Repression is the turning off of gene expression in response to the presence of an end product related compound.

Some genes are regulated in a reversible fashion in response to environmental cues. For instance a cell may or may not synthesize large amounts of a given enzyme in response to the presence of substrates or end products
what are 3 other types of regulation
Constitutive expression. Some genes do not appear to be regulated; they function at the same level under most conditions.

Developmental gene regulation. Some genes undergo “permanent” changes in expression usually tied to overall changes in cell properties.

Cell cycle gene regulation. Some genes are expressed at unique times during the cell cycle.
Molecular Basis of Gene Regulation: Control of Transcription Initiation
Many genes are regulated through modulations of transcription initiation. These systems have been studied by genetics (isolating mutations that alter the regulation), biochemistry (testing purified components for how they act), and structural determinations.
Negative regulation?
involves the inhibition of transcription initiation through the binding of a repressor protein to a specific DNA sequence, the operator, located near or overlapping the promoter. The repressor-operator complex may act by sterically blocking RNA polymerase binding to the promoter. Steric competition acts in lac operon regulation as suggested by the DNA sequence relationships between the promoter and the operator. In most cases, repressor activity is modulated by small molecules (effectors) that either inactivate the repressor (this is found for induction systems like the lac operon) or activate the repressor (for repression) systems.

Bound Repressor Inhibits Transcription
Positive regulation?
involves the activation of transcription initiation as a result of the binding of an activator protein to a DNA sequence located near the promoter. These proteins activate transcription by making a protein-protein contact with RNA polymerase. The DNA binding activity of the activator is modulated by the binding of an effector molecule to the protein. The CAP protein is an activator for lac operon expression. Cyclic AMP (cAMP) is the small effector molecule that regulates CAP binding to its target DNA sequence.

Bound Activator Facilitates Transcription
Regulation at Promoters is both _____ and _____
positive and negative
Protein Recognition of and Binding to Specific DNA Sequences
Repressors and activator proteins are prime examples of proteins that recognize and bind to specific DNA sequences usually by interacting with specific base pairs through their reactive groups in the broad and narrow grooves.
Regulation at a distance
occurs when a protein, that binds some distance from the promoter, regulates the promoter’s activity. This type of regulation occurs in higher organisms but also exists in bacteria (for instance the ara operon as pictured below). One mechanism to explain regulation at a distance invokes DNA looping in which the protein bound some distance cooperatively interacts with a protein bound close to the promoter. The distal sites are sometimes called enhancer elements.
sigma substitution
Different sets of genes are expressed when a different sigma subunit is substituted for the 70 subunit because each type of sigma subunit recognizes a different DNA sequence as a promoter. For instance, when a bacterial cell is stressed by exposure to high temperatures or alcohol, a new sigma factor (32) directs transcription to a new set of promoters
DNA methylation
DNA can be modified by methylation. Methylation of promoter DNA can perturb RNA polymerase binding, probably because the bulky methyl groups inserted in the major groove block the protein’s interaction with reactive groups on the DNA.
DNA rearrangement
Flipping of a DNA segment that contains a promoter will turn on or off a gene.
transcriptional regulators _______ influence the initiation complex
directly
transactivation domains on DNA Binding Protein are ______ for activity
essential
chromatin
active coregulatory molecules
The study of RNA processing has led to three of the most important discoveries in biology:
Eukaryotic genes are interrupted by substantial amounts of “extra” DNA (introns) of unknown function.

RNA can act as an enzyme.

Cells can transfer RNA information into DNA


Almost all of the RNA processing events to be discussed involve theme 4 reactions (phosphoryl transfer reactions).
Overview of Eukaryotic mRNA Processing
Eukaryotic mRNAs undergo a series of major processing steps including: addition of the 5’ CAP, removal of 3’ end sequences, polyA addition to 3’ end, and splicing out of intron RNA.
5’ End Modification: Capping mRNAs
Eukaryotic mRNAs are enzymatically modified to have a 7-methyl guanosine added backward to the 5’ end. This is called a CAP. The CAP is involved in the formation of the translation initiation complex and may act to protect the 5’ end from degradation.
Chemistry of 5’-End Modification Requires Four Enzymes and Regulated by CTD of RNA Pol II
-phosphohydrolase
-guanylyltransferase
-guanine-7-methyltransferase
-2-O-methyltransferase
splicing is?
intron removal

A typical eukaryotic gene has protein coding regions (exons) interrupted by introns (DNA that does not encode a polypeptide chain). Introns compose over 90% of human DNA. After transcription, the intron RNA sequences are precisely removed by a process termed splicing. In most cases, splicing is catalyzed by ribonucleoprotein complexes called spliceosomes. Splicing takes place in two steps. The 5’ exon is released and the 5’ exon is attached to the 3’ exon. In some cases, RNAs are self splicing. These RNAs can act as enzymes and are called ribozymes.
type 1intron self splicing
the 3' OH of guanosine acts as a nucleophile, attacking the phosphate at the 5' splice site

the 3' OH of the 5' exon becomes the nucleophile, completing the reaction
type 2 intron self splicing
the 2' OH of a specific adenosine in the intron acts as a nucleophile, attacking the 5' splice site to form a lariat structure

adenosine in the lariat struct has three phosphdiester bonds

the 3' OH of the 5' exon acts as a nucleophile , completing the reaction
Coordination of splicing by the _______
RNA Pol 2 CTD
3' end processing
Eukaryotic mRNAs are cleaved at precise locations and then 100-200 nucleotide long polyA tails are added. PolyA tails stimulate translation and may play a role in stabilizing mRNAs.
Regulation of splicing and 3' end cleavage
The location of 3’ end cleavage and the nature of splicing events is regulated for some genes. In this way the same gene can encode two different but related mRNAs in two different types of cells giving rise to two different proteins.
transcription cleavage
Ribosomal and tRNAs are typically synthesized as parts of extended transcripts that are then cut at precise locations into functional molecules. One of the nucleases that functions in E. coli tRNA 3’ end cleavage is a ribonucleoprotein complex (RnaseP) in which the RNA is the catalytic subunit.
Base modification in tRNAs
some specific nucleotides in tRNAs are modified

tRNAs have a large number of modified bases
Reverse flow of genetic information
Some viruses that contain RNA as genetic material have the RNA information transferred to DNA after infection. These viruses are called retroviruses. HIV-1 is an example of a retrovirus. The enzyme that catalyzes this process is called reverse transcriptase. Reverse transcriptase can use either an RNA or a DNA molecule as a template. Reverse transcriptase is also found in normal, uninfected cells. Therefore, reverse flow of genetic information may be a “normal” process.
Structures of HIV inhibitors
3'-Azido-2',3'-didedoxy-thymidine (AZT)

2',3'-Dideoxyionsine (DDI)
colinearity of mRNA and Protein Production
Genes encode proteins in a colinear fashion with the orientation of the encoding process being 5’ to 3’ (mRNA) related to the N (amino) terminus to the C (carboxyl) terminus of the polypeptide.
Nucleotide Sequence is Decoded Using Transfer RNAs
The mRNA nucleotide sequence information is decoded into amino acid sequence information by tRNA molecules. tRNAs are short (60-95 nucleotides), highly structured, extensively modified, stable RNAs. Each type of tRNA is capable of carrying a specific amino acid covalently attached to its 3’OH end. The tRNA has a 3 base sequence in a looped structure (the anticodon loop) that base pairs with specific 3 base mRNA sequences. The 5’ end of the anticodon (corresponding to the 3’ end of the message codon) can be more flexible in its base pairing (it “wobbles”).
The Adaptor Hypothesis
Adaptor Function

Converts 3 of 4 letter code into
20 amino acid code

Codon: Triplet of nucleotides for
each amino acid

Translates an open reading frame;
Translation
tRNA structure
Deduced in 1965

Small ss RNAs with 3D structure

25-30 kDa

Contains modified bases and
sugars

pG at 5’

CCA at 3’

G : U pairing

Cloverleaf structures
Cloverleaf structure of tRNA
Pink residues invariant– 4-5 arms

Anticodon arm - 7 unpaired residues

D loop - dihydropyridine

T-C ribothymidine, pseudouridine




All add to folding and adaptive
function

AA is esterified via the carboxyl
group at 2’ or 3’
Aminoacylation of tRNA (Charging)
The loading of each amino acid onto the correct tRNA is an extremely precise process that is catalyzed by an amino acid specific enzyme called aminoacyl-tRNA synthetase. The first step involves activation of the amino acid through adenylation of the carboxyl group. The aminoacyl-tRNA synthetase then picks out the correct tRNA and charges it (transfers the amino acid to the 3’OH end of the tRNA).
Structure of Aminoacyl-tRNA
Amino acyl group is
esterified at 3’ position

Activated tRNA

Proofreading concept

(at amino acyl synthetase
because identify of the aa
is not checked later by the
ribosome)
The Code
The code was determined through genetic deduction studies and direct biochemical analyses; and was confirmed by DNA and protein sequencing and by recombinant DNA experiments.

The code is:
Triplet – 3 consecutive bases correspond to 1 amino acid.
Redundant – each amino acid (except for methionine and tryptophan) is encoded by more than one codon. Two codons that encode the same amino acid are said to be synonymous.
Universal – essentially the same code is used in all organisms.
Biased - different organisms preferentially use different synonymous codons for the same amino acid.

Special codons identify the start and end of a protein coding sequence. AUG is usually the initiating codon. As the initiating codon, AUG encodes the incorporation of a special amino acid, N-formylmethionine. The codons UAA, UAG and UGA are stop or termination or nonsense codons; they do not code for any amino acid in normal cells.
Using the Code to Understand Point Mutations
Mutations that change the mRNA sequence from one synonymous codon to another (in other words, do not change the amino acid that is inserted) are silent mutations – they have no affect on the resulting protein structure. An example would be a UUA (leucine) to UUG (leucine) change.

Mutations that change the mRNA sequence from a codon for one amino acid to a codon for a different amino acid are missense mutations. An example would be UUA (leucine) to UCA (serine).

Mutations that change the mRNA sequence from a codon for an amino acid to a stop codon are nonsense mutations. An example would be UUA (leucine) to UAA (stop). These mutations prematurely terminate protein synthesis at the stop codon site.
reading frames
The mRNA is read in a reading frame, that is as sequential three base codons with no overlap and no punctuation. The “correct” reading frame is set by the initiating codon (AUG).
Mutations that insert or delete 1, 2, 4, 5, etc., but not multiples of 3 nucleotides are frame shift mutations; the reading frame downstream of the mutation is shifted giving an entirely different amino acid sequence. In addition, a stop codon is usually encountered after a short extension.
analyzing reading frames
When looking at a double stranded DNA sequence there are 6 potential reading frames; three going in each direction. A long reading frame uninterrupted by stop codons is called an open reading frame (ORF). The presence of an ORF is suggestive of a protein coding sequence.
Initiation
The initiating amino acid is
N-formylmethionine. This makes sense because protein synthesis involves sequential condensation reactions of carboxyl to amino groups to form peptide bonds and
N-formylmethionine has a blocked amino group.

tRNA is activated, N-formyl gp is added via transformylase
the ribosome
The ribosome is a complex structure composed of two subunits each containing large stable RNAs and several proteins. The ribosome performs two functions. First it is the “organelle” on which translation occurs. Second, the large subunit RNA is the peptidyl transferase enzyme that forms the peptide bonds (see below).
Translation Initiation Complex (for bacteria)
In bacteria, translation initiation involves the base pairing interaction between three different RNAs. The mRNA will form specific base pairs with an RNA found in the small (30S) ribosome subunit. The portion of the mRNA that forms these base pairs immediately precedes the AUG initiating codon and helps distinguish the initiating AUG from other AUG codons. The mRNA AUG base pairs with the fMET-tRNA anticodon loop (CAU). After the initiation complex forms, the 50S ribosome subunit binds.
Translation Initiation Complex in Eukaryotes and Bacteria
In higher organisms there is no base pairing between the mRNA and the ribosomal RNA. Rather it is the first available AUG that acts as an initiating codon. This means that a eukaryotic mRNA typically can initiate translation at only one AUG.
The initiation process is catalyzed by various factors (labeled IF in the figure) and uses GTP as an energy source.

A, aminoacyl site
P, peptidyl site
E, exit site
Recognizing the Eukaryotic AUG
No Shine-Dalgarno

AUG is found via
scanning


40S ribosome uses factors
to tie up the 5’ and 3’
mRNA ends

Linkage facilitates
translation

Poly A binding protein
elongation
The ribosome binds two charged tRNAs; an aminoacyl tRNA with its amino terminus blocked to the “P” site, and an aminoacyl tRNA with a free amino group to the “A” site. The tRNAs are held to consecutive 3 base codons on the mRNA; the tRNAs are specifically base paired through their anticodon loops to the mRNA and it is through this process that they are “chosen.” A transpeptidation event (catalyzed by an RNA in the large subunit) occurs that condenses the carboxyl group from the first amino acid to the amino group of the second amino acid. The first tRNA (with no attached amino acid) is ejected, the ribosome translocates three bases down the message to the next in-frame codon, and the tRNA carrying the peptide shifts position on the ribosome to the P site. A new charged tRNA comes in to the A site as dictated by the base pairing to the next codon.

Incoming acyl tRNA binds
to Tu-GTP then to the A site

Binding leads to release of
Tu-GDP

Tu-GTP is regenerated

Peptide bond is formed when
A and P sites are occupied

Alpha amino gp of incoming
aa at A acts as a nucleophile
at the carboxyl gp at P

Creates a dipeptidyl tRNA at A

Causes the release of aa from
tRNA at P

tRNAs shift towards E and P sites

Ribosome translocates 5’ to 3’

Shifts the anticodon of the
dipeptidyl tRNA to the P site and
the deacylated tRNA to the E site

Requires energy

Now positioned for next aa

Proofreading

Speed is balanced by fidelity

2 GTPs are utilized per codon
The Large Ribosome Subunit RNA is the Peptidyl Transferase
The molecular structure of the large ribosomal subunit bound to a peptidyl transferase active site inhibitor was recently determined (Nissen et al. (2000) Science vol. 289, 920). This structure shows that the bulk of the subunit is RNA (with small patches of protein) and that the peptidyl transferase active site (where the inhibitor is bound) is made up of RNA alone. This is good evidence that RNA is the enzyme in this case. Next is a copy of the picture of the ribosome.
the 50S subunit-RNA is responsible for ?
peptidyl transferase activity
chain termination
Termination of protein chain elongation occurs in response to a termination codon moving in frame into the A site. A release factor (RF1 or RF2 ) binds to the A site. This leads to hydrolysis of the nascent peptide – tRNA linkage. The mRNA, deacylated tRNA, and release factor leave the ribosome and the ribosome dissociates to its subunits.

Energy: 4 NTPs for each peptide bond (addition plus proofreading)

Two specific amino acids added
Protein modification - final step
Proteins undergo substantial covalent modifications after synthesis. The N-terminus of the primary translation product is modified; either the formyl group or the entire formylmethionine is removed. Proteins that are transported across membranes frequently have a short N-terminal sequence removed. Some proteins are subjected to other forms of covalent modification such as glycosylation or hydroxylation at specific residues. A very important type of protein processing involves proteolytic cleavage at specific sites. For instance several proteins encoded by an HIV-1 mRNA are actually part of a very large polypeptide that is cleaved into functional sections by a virus specific protease. It is this enzyme that is targeted by the anti HIV-1 protease inhibitors.
Regulation A,B,C
A Direct regulation of translation initiation by proteins.
The translation of some genes is regulated by the binding of a protein to the translation initiation signals. In eukaryotes translation is frequently regulated by proteins binding to the 3’ end sequence of the mRNA and then interacting with the 5’ end.
B Antisense RNAs.
The translation of some mRNAs is blocked by the synthesis of antisense RNAs that base pair with the mRNA in the region of the translation initiation signals. Remember that these signals must be free to base pair with the ribosomal RNA and the fMet-tRNA anticodon loop.
C Transcription termination vs. elongation.
The expression of some genes is determined through regulating the termination of transcription that can occur at a site preceding the gene. Regulation of transcription termination can occur through a complex coupling to translation. See next figures describing the regulation of the trp operon system.
Trp Operon Leader Sequence
Co-transcription and translation

Regulatory sequences located between
DNA sequencing
DNA sequencing allows us to determine the precise structure of genes. The techniques used involve the generation of nested families of molecules all of which have one end (5’) in common. At the 3’ end are four families: one family ends with A residues, one ends with G’s, one ends with C’s, and one ends with T’s. These molecules are fractionated according to size by high resolution polyacrylamide gel electrophoresis.
The most common method for generating a nested family of fragments utilizes dideoxynucleotide analogues as substrates of DNA polymerase action. A dideoxynucleotide has an H in place of an OH at the 3’ position thus when incorporated, it blocks all further elongation. Recall that the elongating 3’OH plays a critical role in the theme 4 reaction catalysis. By mixing in a small amount of dideoxy A in a DNA polymerization reaction, one generates a family of DNA molecules all with one end in common (as defined by the original primer) and the other end defined by the incorporation of dideoxy A residues at different sites opposite the T residues of the template. The same approach is taken with dideoxy G, C and T; and the four families of products are analyzed together by high resolution poyacrylamide gel electrophoresis.
Dideoxies block _____
elongation
Restriction enzymes
Type II restriction enzymes bind to (theme 2) and cut (theme 4) specific DNA sequences. The DNA target of different restriction enzymes is typically a symmetrical sequence of 4, 5, 6 or 8 base pairs. Each type of enzyme has a unique target specificity and a unique mode of cutting the DNA, although all of them generate molecules containing a 5’ phosphate and a 3’ OH. A given type of restriction enzyme cuts any given specific DNA molecule into a characteristic (and inherited) pattern of fragments that can be analyzed by gel electrophoresis.
There are several different uses for restriction enzymes. Restriction enzyme cutting of DNAs is typically the first step in DNA cloning. Since the cleavage pattern is inherited, it can be used to diagnose the presence of mutant genes underlying genetic diseases or it can be used in forensics to identify the source of a tissue sample.
DNA ligation
DNA ligase links a 5’ PO4 to a 3’ OH group to form a phosphodiester bond (theme 4 chemistry). Ligase functions inside the cell to seal the nicks in lagging strand synthesis. We use ligase in the research lab to link together recombinant DNA molecules.
Mechanism of DNA ligase Action
1. Adenylylation of DNA ligase
2. Activation of 5' phosphate in nick
3. Displacement of AMP seals nick
cloning
the generation of recombinant dna

Recombinant DNA molecules are formed by inserting “foreign” DNA into a “vector” or “vehicle” DNA molecule. Vector DNA molecules typically have the following features: an origin of replication suitable for the host of choice, a function that allows one to select cells containing the vector, and an easy means for inserting the foreign DNA (restriction enzyme cut sites). A typical recombinant DNA experiment involves cutting the foreign and vector DNAs with the same restriction enzyme, ligating the two molecules together using DNA ligase, introducing the recombinant DNAs into the host cell, selecting for the presence of the vector during propagation of the host cell, and examining the structure of the recombinant DNA’s.
Recombinant DNA experiments allow the amplification and purification of specific DNA fragments for further study. Cloning is also an essential component of foreign gene expression systems. In these cases the vector has been constructed to provide the required gene expression signals (promoter and translation initiation signals) immediately next to the cloning site.
synthetic DNA and in vitro mutagenesis
Synthetic chemical procedures have been developed that allow synthesis of defined DNA sequences. Usually these molecules are limited in length (no more than a hundred bases). These techniques can be used to synthesize entire synthetic genes or DNA regulatory elements, which when cloned allow the introduction of novel genes into cells. This procedure allows a definitive scientific test of molecular biological concepts such as the code, promoter activity, translation initiation signals, etc., and confirms that DNA is the genetic material.
In a more limited approach, small synthetic DNA molecules can be used to introduce specific or random point mutations into recombinant DNA molecules.
An important application of synthetic DNA chemistry is in the production of defined hybridization probes and/or defined primers for DNA polymerization assays.
Polymerase Chain Reaction (PCR)
PCR involves multiple rounds of amplification of short DNA segments that are defined by two primers that “face each other.” PCR uses DNA polymerase from a thermophylic microorganism so that the enzyme is stable for the many rounds of heat denaturation. PCR can be used to detect and amplify for analysis very rare sequences.