CHEM 430 UNIT 4

Overall transcription reaction

(RNA)n + NTP <-> (RNA)n+1 + PPi Identical to DNA polymerase chem (3'OH nucleophile attacks alpha phosphate 2Mg2+ ions stabilize side chains in active site)

2nd step in prokaryotic transcription

- 10-12 BPs of dsDNA melted out to expose template DNA strand - Closed complex -> open promoter complex - Formation of a transcription bubble around melted out site - RNAP can access template strand

Wobble Rules

- 5' G @ anticodon base pairs with 3' codon (wobbles C/U) - 5' U @ anticodon base pairs with 3' codon (wobbles A/G) The 3 position of the codon is what wobbles, the anticodon can only code for one letter

DNA Polymerase I (DNA Pol I)

- 5'->3' synthesis of DNA activity - Two exonuclease activities - "slow", low processivity

How does 3rd position wobbling benefit the cell?

- Minimizes the number of tRNAs required in the cell (a single tRNA can "cover" two triplet codons if they only differ in the wobble position) - Allows for some degree of mutation at DNA level without changing the protein product primary sequence

Polypeptide synthesis Overview

- Proceeds from N -> C terminus - Chain elongation occurs by transpeptidation (synthesis of a peptide bond) - Ribosomes read mRNA in the 5'->3' direction

3rd step in prokaryotic transcription (elongation)

- RNA synthesis proceeds 5'->3' - Incorporates NMP 1 at a time through series of nucleophilic attacks (3'OH and a phosphate) - Transcription bubble is maintained throughout - DNA-RNA hybrid maintained through complementary base paring - Completely processive (if elongation complex (DNA, RNA, RNAP) is disrupted, transcription is irreversibly terminated

Eukaryotic RNA Splicing Reaction

- Removal of introns catalyzed by RNA (RNA acts as reaction catalyst) - Pre-mRNA contains a 5' and 3' splice site (dictates where the intron is relative to the exon) - these are conserved sequences found at the splice junctions of all eukaryotic pre-mRNAs - Spliceosome: complex of pre-mRNA, snRNA, and proteins. - Splicing is the results of two transesterification reactions

Using the DNA footprinting technique, which of the following were/was identified as binding site(s) for s factors?

-10 and -35

DNA replication cannot occur from 3' to 5' because...

-OH group is on the 3' end and is necessary for nucleophilic attack of the dNTP NOT dNMP

Translocation in translation

-completes the cycle -ribosome advances three nucleotides along the mRNA in the 5' to 3' direction -concurrently, the uncharged tRNA from the P site is expelled and the peptidly-tRNA from the A site moves into the P site -ribosome has an empty A site ready for the entry of the aminoacyl-tRNA corresponding to the next codon

Exonuclease activity of DNA Pol I (2 kinds)

1) 3'->5' Proofreading. Synthesis is 5'->3' so proofreading is 3'->5' (DNA Pol I removes any incorrect nucleotides as DNA is synthesized) 2) 5'->3' RNA primer removal (Nick translation), removes RNA primer (NMPs) and simultaneously fills in with DNA dNTPs Removes RNA primer and fills in with dNMPs using dNTPs as substrates

Ribosome Binding Site

1) A/Aminoacyl site: Accommodates the incoming aminoacyl-tRNA 2) P/peptidyl site: accommodates the peptidyl-tRNA, the tRNA to which the nascent (growing) peptide chain is attached 3) E/Exit site: Accommodates the deacylated tRNA that is about to EXIT the ribosome

The charging reaction (attachment of an AA to the correct tRNA by amino-acyl synthetase)

1) Alpha COO- from nucleophile acts as AA to attack alpha phosphoryl 2) Off comes PPi (Amino-acyl AMP intermediate formed) 3) 3' -OH attacks carbonyl (AMP released) 4) Aminoacyl-tRNA formed

4th step in prokaryotic transcription (termination)

1) Rho-independent (intrinsic): Sequences in DNA are expressed as RNA which form structures that cause termination of elongation (ex.) RNA hairpin loop) 2) Rho-dependent (extrinsic): protein (termination factor) binds to growing RNA and causes physical dissociation of RNAP and DNA (ex.) rho protein)

Release Factors (termination of transcription)

1) Stop codon brings in release factor (NOT tRNA) 2) Release factor binds to A site, stimulates cleavage (de-Acetyl tRNA released) 3) RFs dissociate from A site, tRNA is released 4) 50S and 30S subunits dissociate

Sources of fidelity to minimize errors in DNA replication

1) dNTP conc'n in cell is kept low 2) Bind correct dNTP via AT:GC base pairing (ensure proper geometry at the active site, then incorporate dNMP) 3) Proof-reading mechanism

Initiation step of translation

1) dissociation of ribosome (50S and 30S) 2) binding of mRNA (IFs interact w/ mRNA + ribosome to form initiation complex) 3) Binding of 50S subunit, TFs come off (GTP dependent) Now tRNA that corresponds to coding region needs to be brought into the A site

Assume that a culture of E. coli was grown for approximately 50 generations in 15N (provided in the medium in the ammonium ion), which is a heavy isotope of nitrogen. You extract the DNA from the culture, and it has a density of 1.723 gm/cm3 (water = 1.00 gm/cm3). From the literature, you determine that DNA containing only the common form of nitrogen, 14N, has a density of 1.700 gm/cm3. Bacteria from the 15N culture were washed in buffer and transferred to 14N medium for one generation immediately after which the DNA was extracted and its density determined. What would be the expected approximate density of the extracted DNA?

1.712 gm/cm3 15N cultured in 14N yields 1.71 g.cm^3

Assume you replicate the Meselson-Stahl experiment. After switching E. coli cells to 14N media, you collect DNA and call these cells, G1. 100% of G1 DNA is 15N/14N hybrid DNA. If you were to allow cells to continue to replicate semiconservatively, what fraction of G2 DNA would be 15N/14N? What fraction of DNA would be 15N/14N at G3?

1/2; 1/4

Assume you replicate the Meselson-Stahl experiment. After switching E. coli cells to 14N media, you collect DNA and call these cells, G1. 50% of G1 DNA is 15N/14N hybrid DNA. If DNA Replication were conservative, what fraction of G2 DNA would be 15N/15N? What fraction of DNA would be 15N/15N at G3?

1/4; 1/8

The Shine-Dalgarno sequence of mRNA base pairs with the _ rRNA within the prokaryote _ ribosomal subunit.

16S; 30S

Number of Pol III enzymes

2 Pol III enzymes per replication fork in the replisome

How many origins of replication do eukaryotic chromosomes have?

3

The anticodon of tRNA is made up of _ bases.

3 (so is the codon)

Codon (triplet code)

3 base sequence that specifies a single amino acid 4 possible bases, 20 different amino acids Each triplet code has 4^3 = 64 possible sequences

The prokaryotic ribosome contains _ subunits. The eukaryotic ribosome contains _ subunits.

30S and 50S; 40S and 60S

DNA polymerase moves in the ____ to ____ direction.

5' -> 3' to remove RNA and synthesize (fill in) missing DNA

A coding strand of DNA is as follows: 5' CGCTATAGCGTTT 3'. What is the mRNA transcript?

5' CGCUAUACGCUUU 3' mRNA transcript is the same as coding/sense strand (T/U) and complementary to the non-coding/antisense strand

Which base position is LEAST discriminating in the bases that it can pair with as described by the wobble hypothesis?

5' end of the tRNA anticodon or 3' end of mRNA codon

Subunits of the prokaryotic ribosome

50S (large) and 30S (small) Total size = 70S (S values aren't additive) 66% RNA by mass 34% protein by mass - rRNAs play a critical role in both the structure of RNA as well as catalysis

5' 7-methyl guanosine cap

7-methy guanosine cap added to 5' end of eukaryotic mRNA - 5'-5' triphosphate bridge (chemically makrs 5' end of mRNA)

In E. coli, _ RNA-DNA base pairs are maintained and about ____ base pairs of single-stranded DNA are maintained in the transcription bubble.

8;17

tRNA interacts with ribosomes at one of three sites: the E site, the P site, and the _ site.

A

Which is NOT required in the prokaryotic transcription initiation complex? a primer RNA polymerase initiation factor a ribonucleotide triphosphate (NTP)

A primer

Where might the a polypeptide chain be located in both the prokaryotic and eukaryotic ribosome?

A site or P site

The tRNA 3' → 5' anticodon loop that recognizes the 5' → 3' codon UCC could have the sequence

AGG

A full catalytic cycle of an aminoacyl-tRNA synthetase generates _ as a product.

AMP

Correct vs. Incorrect base pairing

AT and GC paired together (consistent spatial geometry (correct for phosphodiester bond synthesis)) Poorly/incorrectly paired bases (misaligned geometry via hydrogen bonding)

Approximately how many more molecules of ATP are produced under aerobic conditions (PDC, TCA, and ETC) than under anaerobic conditions (glycolysis)

About 30 Anaerobic conditions: 2 ATP Aerobic conditions: 32-34 ATP

DNA Polymerase Enzyme Mechanisms

Acid-Base catalysis (3' OH) Metal-ion Catalysis (Mg2+ stabilization)

The peptidyletransferase mechanism involves

Acid-Base chem - Polypeptide chain increases by 1 AA each time an amino-acyl tRNA is brought into the A site

When is the poly A tail and 5' methylguanosine cap added to the mRNA?

After transcription to protect the mRNA from degradation and give it the ability to enter the nucleusf

What does DNA look like when DNA Pol I is finished?

All DNA now, no RNA 1 nick (okazaki fragment) left that needs to be filled in

Which RNA molecule(s) bind to the ribosome? tRNA rRNA mRNA all the above

All of the above

Which functional group does the -OH of the tRNA acceptor stem nucleophilically attack during the charging reaction?

Alpha carboxyl group of the aminoacyl AMP Intermediate

Peptidyl transferase connects the carboxylate group of one amino acid to the amino group of an incoming amino acid. What type of linkage is created in this peptide (covalent) bond?

Amide (N-C)

Aminoacyl-tRNA synthetase

An enzyme that joins each amino acid to the appropriate tRNA. Aminoacid + ATP -> Aminoacyl-tRNA(binds to 3' OH acceptor stem) + AMP + PPi

Nature of the genetic code

Arrangement of the code table is nonrandom (most synonyms occupy the same box) Genetic code is universal (can be applied to all organisms)

What helps select the translation initiation site?

Base pairing between mRNA and the 16S ribosome Ex.) Lac operon - SD sequence upstream of AUG tells ribosome which AUG to start translation at - 5' end of SD Region interacts with 3' region of 16S rRNA (structural component of ribosome) - Positions mRNA such that next AUG downstream of SD sequence is the initiation codon

At what point during transcription does the sigma factor dissociate from the RNA polymerase?

Before the elongation phase Binds to promoter to recognize start site then dissociates

Which directional model of replication does DNA synthesis use?

Bidirectional in most prokaryotes and ALL eukaryotes. Always synthesized 5'->3' Nucleotides can only be added to the 3' OH end of the molecule

The ribosome:

Binds mRNA such that codons can be read Includes specific binding sites for tRNA molecules Mediates interactions between non-ribosomal proteins that help facilitate protein synthesis Catalyze polypeptide formation Undergoes movement so that it can translate sequential codons (Has large and small subunit)

How are amino acids covalently attached to tRNAs?

By Aminoacyl-tRNA synthetases = charging reaction Highly accurate process Net Reaction: Amino acid + tRNA + ATP -> aminoacyl-tRNA + AMP + PPi (goes to 2Pi) - Cell possesses 20 different AA synthetases (1 for each of the 20 amino acids)

RNA is a highly dynamic biomolecule in that it

Can fold and hydrogen bond with itself, DNA, proteins, or small molecules to adopt largely modified tertiary structures.

After the first round of replication in the Meselson-Stahl experiment, scientists were able to conclude that DNA replication was ...

Cannot conclude after 1st round (either semi-conservative or dispersive) Could conclude semi-conservative after second round of replication

Splicing Reaction in Eukaryotes

Catalytic RNAs bind to 5' splice site (E1) and 3' splice site (E3) Branch point 2'-OH attacks E1 (5') phosphate, now have 3' OH from exon 1 - This E1 3' OH attacks E2 phosphate Lariat intron is excised

DNA Ligase

Catalyzes the formation of missing phosphodiester bond (seals last nick in DNA/fixes Okazaki fragments)

DNA Polymerase III (DNA Pol III)

Catalyzes the synthesis of leading and lagging strands (responsible for bulk of DNA synthesis) - Only proofreading (3'->5') exonuclease activity (no 5'->3' exonuclease activity/removal of primers and filling in) - Fast and highly processive - E. coli genome = 4.7 million base pairs

tRNA must be _ before binding to the ribosome to allow for translation to occur.

Charged with an amino acid

During translation, the proper tRNA is selected through specific ______ interactions

Codon-anticodon (antiparallel base pairing between codon and anticodon). Specific watson-base pairing between first 2 regions of the codon, 3rd position can wobble

RNAP binds to

Consensus Sequences (-10 and -35 in proks)

tRNA structure part II

Contain modified bases and some non-Watson-Crick base pairing which helps bring structure to the tRNA molecule - Have a complex tertiary structure (base stacking and base pairing between helical stems) - Base pairing of stems = secondary structure (remember 3' CCA acceptor stem end and anticodon region)

Leading strand of DNA has _____ replication. The lagging strand has _____ replication.

Continuous; discontinuous Both synthesized 5' -> 3' Leading strand lines up with 3' -> 5' end of template DNA (synthesizes in same direction as parent DNA), lagging strand synthesizes on 5' -> 3' template strand (synthesizes in opposite direction of the parent DNA, R -> L)

Strands in transcription

DNA Non-template (coding strand): tells you what AA sequence this gene codes for. Same sequence as RNA transcript just with T instead of U DNA Template Strand: complementary to coding strand/RNA transcript RNA transcript: complementary to template strand, same as coding besides U/T

Which of the following enzyme(s) possesses 5' → 3' synthesis activity and 5' → 3' exonuclease activity?

DNA Pol I

Which of the following enzyme(s) possesses 5' → 3' synthesis activity and 3' → 5' exonuclease activity?

DNA Pol I DNA Pol III (does not have 5'->3' exonuclease (RNA primer removal) activity)

Which of the following is believed to be responsible for removing the primer and filling in the gaps left behind during replication?

DNA polymerase I

DNA is replicated by enzymes known as ____ they are _____ dependent

DNA polymerases DNA

What do sigma factors bind to during RNA synthesis? DNA promoters and RNA polymerase RNA promoters and DNA DNA and RNA transcription factors

DNA promoters and RNA polymerase

How many template strands are required for DNA replication vs. transcription?

DNA replication: 2 template strands Transcription: 1 template strand

Transcription in eukaryotes

Defined nucleus (where transcription occurs) RNA processing (doesn't happen in proks) Compartmentalization (transcription occurs in a specific compartment)

Alternative Splicing

Different combos of exons from the same eukaryotic gene can be processed into different mRNAs and then undergo translation into diff. proteins in a tissue specific manner Ex.) a-Tropomyosin gene is specific to 7 different tissue types - Single gene can be spliced by diff. tissue types to give tissue specificity to final protein product

RNA is _____ involved in protein synthesis whereas DNA is ______ involved in protein synthesis

Directly Indirectly

The net reaction of glutamic acid attachment to tRNA is:

E + tRNA + ATP -> aminoacyl-tRNA + AMP + PPi (Charging reaction)

The genetic code is degenerate

Each codon encodes only 1 amino acid An Amino Acid can be encoded by more than one codon UAG, UAA, UGA = stop codons AUG = start codon

Dispersive model of DNA replication

Each strand of both daughter molecules contain a mixture of old and newly synthesized DNA G1: 50% old, 50% new Proportion of new DNA/old DNA increases with replication but old DNA (15N) never completely goes away

Prokaryotic transcription overview

Enxyme = RNAP (RNA Polymerase) - requires a DNA demplate (only 1 RNAP) - Large, multienzyme - Core enzyme: a2BB'w (catalytically inactive, synthesizes the RNA polymer) - Holoenzyme: a2BB'wo (has sigma - binds promoter) - Enzyme requires Mg2+

Primosome

Enzymatic component of the replisome that synthesizes short RNA primers (about 20 nucleotides in length) from nothing

Eukaryotic mRNA needs to be processed

Euk. RNA contains: 5' 7-methyl guanosine cap Euk. RNA possesses a: Poly A tail Euk. RNA contains: exons (code for protein) and introns (get spliced out) The euk. RNA splicing reaction

Natural error rate in DNA replication

Fairly high 1 mistake/1000 BPs

True or False: Wobble allows a codon in mRNA to hybridize to more than one tRNA.

False Wobble allows anticodon in tRNA to hybridize with different codons in mRNA. Allows a tRNA /amino acid to hybridize with more than on codon in mRNA

True or False: DNA replication is a catabolic pathway

False! Replication is an anabolic pathway (building polymer of nucleic acids from individual parts)

True/False: Ribosomes are mostly made of protein.

False! They are mostly made of rRNA

True or False: The Wobble base is the third base (5' to 3') of the anticodon.

False. First base (5' to 3') of anticodon and 3rd base (5'-3') of codon)

True or False: Three different codons code for the start of protein synthesis.

False. Only one (AUG)

The Shine-Dalgarno sequence helps recruit RNA polymerase to DNA in initiating transcription.

False. The SD sequence helps recruit the ribosome to DNA in initiating translation

Which cluster of terms accurately reflects the nature of DNA replication in prokaryotes?

Fixed point of initiation, bidirectional, semiconservative (same as eukaryotes except eukaryotes have multiple origins of replication and prokaryotes only have one)

Semi-conservative model of DNA Replication

G1: 100% 15N/14N G2: 50% 15N/14N, 50% 14N/14N G3: 25% 15N/14N, 75% 14N/14N 15N/14N decreases in proportion to 14N/14N but NEVER disappears

Conservative model of DNA Replication

G1: 50% 15N/15N, 50% 14N/14N G2: 25% 15N/15N, 75% 14N/14N G3: 1/8 15N/15N, 7/8 14N/14N NEVER 14N/15N

Once again you replicate the Meselson-Stahl experiment; however, in this instance assume DNA Replication is semi-conservative. Which statements below are true? G0=cells in 15N media; G1=cells that have been shifted to 14N.

G2 DNA would consist of 1/2 15N/14N hybrid DNA molecules and 1/2 14N/14N DNA duplexes G2 DNA would produce 2 bands. One band consistent with 15N/14N DNA and one consistent with 14N/14N DNA (equal quantities)

Strong prokaryotic promoters: bind tightly to the transcription factors. have s factors that are larger, more stable proteins. generally result in a higher rate of transcription. are less common in prokaryotes.

Generally result in a higher rate of transcription

Translation is the process by which:

Genetic information that has been passed to RNA from DNA is ultimately expressed as a protein. Series of chemical events that catalyze the synthesis of polypeptides from mRNA (Anabolic Process)

Weak prokaryotic promoters: can bind to different transcription factors. have a DNA sequence that is significantly different from that of other common promotors. can be easily bound by sigma factors. give weak DNA footprinting signals.

Have a DNA sequence that is significantly different from that of other common promoters

Controls in Meselson-Stahl Experiment

Heavy isotope of nitrogen (N15) Bacteria can make all N-bases, therefore, all DNA will be heavy DNA Density = 1.724, moves to bottom of tube

RNAP Holoenzyme (has sigma) binds to a promoter

In bacteria: RNAP holoenzyme binds to -10 and -35 (upstream of +1 site) Consensus sequences are evolutionarily preserved "similar" sequences upstream of +1

In which direction is mRNA synthesized by RNA polymerase?

In the 5'->3' direction

N-formylmethionine (fMet)

Initiation amino acid (interacts with start codon) Has amide bond (on N-terminal of a polypeptide) - "Formyl Cap" functional group as as a cap preventing the alpha-amino group from acting as a nucleophile (N-methionine is rendered inactive due to formyl group)

In the ETC, the electron from NADH moves from ______ the mitochondria through the protein complexes to the ______ of the mitochondria

Inside; inside

rRNA (ribosomal RNA)

Integral STRUCTURAL component of the ribosome Makes up ~ 66% of the ribosome mass Doesn't encode info that is used to translate into a protein (they are structural)

Eukaryotic Transcription

Intrinsic factors (IFs) and TATA binding protein (TBP) help recruit RNA pol II to transcription site - Activator proteins interact with enhancer sequences

RNA primase provides RNA primers which are needed for DNA synthesis

Leading strand usually requires one RNA primer Lagging strand requires multiple RNA primers RNA primers need to be removed. Okazaki fragments need to be ligated.

How is the genetic code nonoverlapping?

Letters (nucleotides) cannot belong to more than 1 codon "read" sequentially in units of 3, no overlap between codons

Controls in Meslson-Stahl Experiment

Light isotope of nitrogen (N14) Bacteria can make all N-bases, therefore, all DNA will be light DNA Density = 1.710, stays at top of tube

Helicase (DNAB protein)

Locally "melts" dsDNA -> ssDNA so Pol III can access information in template strands

Your friend Grimes doesn't happen to eat a whole lot of avocados, nuts, tofu, etc. which are rich in magnesium. In the unlikely scenario that she had ZERO magnesium ions in her body, how might this affect DNA replication?

Magnesium ions are necessary for metal ion catalysis in DNA polymerase and other enzymes

On the lagging strand of each replication fork, ______ RNA primers are required for DNA polymerase activity to occur.

Multiple Leading strand - 1 RNA primer

Consider an in vitro experiment in which all components needed for protein synthesis are present. If tRNAs charged with radioactively labeled amino acids are added, over time the radioactivity would be located in which of the following components?

Nascent (growing) protein tRNA adds amino acids to the growing protein

Transcription in prokaryotes

No Nucleus Transcription occurs in the cytosol dsDNA -> ssDNA -> protein

The genetic code is commaless

No additional nucleotide separating 2 codons

A given sequence of bases in an mRNA will code for one and only one sequence of amino acids in a polypeptide, if the reading frame is specified. From a given sequence of amino acid residues in a protein, can you predict the base sequence of the unique mRNA that coded it?

No. A single amino acid can be coded for by more than one codon (sequence of mRNA)

If a mutation were to occur on polA which encodes for DNA pol I and remove its 5' 3' polymerase and 3' 5' exonuclease activity, we would expect the bacteria to... a) have decreased 5' to 3' polymerase activity, thus severely limiting its ability to reproduce b) be unable to 'fix' mistakes in DNA and thus we would see significantly more mutations c) Both A and B d) None of the above

None of the above

Once again you replicate the Meselson-Stahl experiment; however, in this instance assume DNA Replication is dispersive. Which statements below are true? G0=cells in 15N media; G1=cells that have been shifted to 14N. G1 DNA would produce two bands after ultracentrifugation. One consistent with 15N/14N hybrid DNA and one consistent with 14N/14N DNA G2 DNA would consist of 1/4 15N/14N hybrid DNA molecules and 3/4 14N/14N DNA duplexes G2 DNA would consist of 1/2 15N/14N hybrid DNA molecules and 1/2 14N/14N DNA duplexes G2 DNA would produce two bands. A faint band consistent with 15N/14N hybrid DNA and a more intense band consistent with 14N/14N DNA None of these

None of these Dispersive is always 14N/15N band, Just gets farther to the left bc more 14N than 15N

Result of Experiment 1 Meselson-Stahl (grow cells in N15 and allow to replicate, then shift cells to media containing N14)

One density peak at 1.717 (between density of 14N and 15N and only one peak) - must be semiconservative. Each DNA molecule has one strand heavy DNA and one strand light

Result of Experiment 2 Meselson-Stahl (allow cells to replicate a second time in media containing N14), is the semi-conservative or dispersive model correct?

One peak at 14N/14N and one peak at 15N/14N - must be semiconservative. If dispersive, you would see ONE peak around 15N/14N that would have shifted slightly to the left from the previous peak (more 14N DNA)

DNA polymerase requires an ________

Origin of replication

In the initiation phase of translation the tRNA is in the

P site

What drives the anabolic process of phosphodiester bond formation?

PPi -> 2 mol of Pi (exergonic reaction) Pyrophosphate side-product gets converted into 2 moles of inorganic phosphate

Transcription in Eukaryotes overview

Polymerases: 1) RNA Pol I (rRNA) 2) RNA Pol II (mRNA) 3) RNA Pol III (tRNA, snRNA, 5S rRNA) Consensus sequences: - different than the -10 and -35 elements in proks. - TATA box - GC boc - Enhancers or silencers Eukaryotic transcription requires transcription factors (proteins) in addition to RNA polymerase

RNA is typically more susceptible to backbone hydrolysis than DNA because of the

Presence of a 2' OH group

1st step in prokaryotic transcription

Promoter binding (sigma subunit binds) and interacts with both the bases and sugar phosphate backbone to form the "closed complex" Still dsDNA

Organization of genes in prokaryotes

Protein-encoding genes are arranged in tandem along a single strand and are transcribed together as a SINGLE unit = operon - Genes are transcribed as 1 long polycistronic - Genes are not transcribed individually, they are transcribed as 1 long mRNA (single mRNA contains genetic info for translation of multiple indiv. genes)

Enhancers are transcriptional regulatory sequences that function by enhancing the activity of...

RNA polymerase at a single promoter site

In bacterial transcription, the sigma factor dissociates during elongation. How would the activity of the polymerase and transcription elongation be affected if the sigma factor remained bound to the holoenzyme after initiation?

RNA polymerase would be unable to continue down the template. Dissociation of the sigma factor increases the affinity of the holoenzyme for general DNA, allowing the enzyme to move along the template

Prokaryotic transcription could terminate by: association of a sigma factor with the DNA. RNA with GC stem loop structures. a G-rich region on the DNA. RNA polymerase without the elongation factor.

RNA with GC stem loop structures (hairpin loop) or rho-protein presence

DNA Foot Printing (sigma subunit of RNAP binds tightly to conserved sequences in the promoter region)

Radio-labeled with 32P RNAP binds to experimental (also have control with no RNAP) - Produces a pool of cleavage products of diff. lengths on gel - Where RNAP is bound it protects the DNA (DNA cannot cleave here), no cleavage products (this area appears blank on a gel)

The 5' → 3' exonuclease activity of E. coli DNA polymerase I is involved in:

Removal of RNA primers by nick translation. Polymerase 1 not 3! 3'->5' exonuclease activity is proofreading

Bidrectional DNA replication

Replication from the origin can move in 2 opposite directions. Two replication forks moving in two opposite directions relative to the origin.

Unidirectional DNA replication

Replication from the origin can move in one direction. One replication fork moving in one direction relative to the origin. 5' -> 3'

Where does translation occur?

Ribosomes in the cytoplasm (2/3 structural RNA and 1/3 protein)

mRNA (messenger RNA)

Sequence that directs protein synthesis (translational)

The difference between polycistronic DNA and alternative splicing is...

Several proteins can be produced from one mRNA transcript from polycistronic DNA

Okazaki fragments

Short fragments of DNA on the lagging strand

Translocation (Translation)

Starting: have peptidyl tRNA, A site is empty 1) AA is brought into A site by tRNA (binding of specific AA-tRNA to A site) 2) Peptide bond formation between new AA from tRNA and existing peptide chain, chain transfer from peptidyl-tRNA to aminoacyl-tRNA 3) Translocation of peptidyl-tRNA from A site to P site. Ribosome moves 1 codon to the right and the now uncharged tRNA (still bound to first codon) moves from P site to E site (then tRNA exits from E site)

DNA bands in Semi-conservative model

Starting: one heavy band at 15N/15N Round 1: One dark band at 15N/14N (slightly father left than previous band) Round 2: One band at 14N/14N, one band at 15N/14N. Band at 14N/14N is farther left Round 3: One band at 14N/14N (much darker and thicker), one band at 15N/14N (has gotten much fainter)

What is the primary difference between strong and weak bacterial promoters? How does transcription differ from the two types of promoters?

Strong bacterial promoters differ slightly from conserved sequences at the -35 and -10 boxes. Weak promoters may contain several base pair changes. The closer the -35 and -10 boxes are to the consensus sequences, the stronger the association between RNA polymerase and the promoter, so transcription is more likely to occur (and at a faster rate) with strong promoters

tRNA (transfer RNA)

Structurally compact RNAs that deliver amino acids to the ribosome during translation

The ___ strand of DNA is transcribed into mRNA.

Template (non-coding)

Chain termination Translation

Termination codons (UAA, UGA, UAG) have no corresponding tRNAs - These codons are recognized by RELEASE FACTORS (not tRNAs), protein component stimulates the release of the peptide from peptidyl-tRNA - Binding of RF, rather than aminoacyl-tRNA, to a termination codon induces the ribosomal peptidyl-transferase to transfer the peptidyl group to water

In prokaryotic transcription, termination is dependent on:

Termination protein factor (rho-dependent) Hairpin loop on mRNA causes dissociation (rho-independent)

The discontinuous aspect of replication of DNA in vivo is caused by ____.

The 5' to 3' polarity restriction

How does Aminoacyl-tRNA synthetase know which amino acid to add to tRNA?

The anticodon region and other regions encode specificity for the enzyme - The synthetic oligonucleotide will be charged by the correct Aminoacyl-tRNA synthetase and the correct AA will be added to the 3' acceptor stem

Which group will be attacked by the nucleophilic amino group of the next amino acid brought into the ribosome?

The carboxyl C of the nearest AA to the ribosome

Which model of DNA replication could be discarded after Meselson-Stahl Experiment 1?

The conservative model Would have seen 2 peaks (one at heavy 15N density and one at light 14N density)

Why does the initiator tRNA bind in the P site rather than the A site of the ribosome?

The initiator tRNA charged with fMet begins the protein sequence. Because Met is the first amino acid, it must bind directly to the P site so that a second tRNA can bind to the A site and the first peptide bond can be formed (AA is A site nucleophilically attacks C of fMet in P site)

What will happen to the intensity of the two peaks as replication is allowed to continue in media containing N14?

The light DNA peak will increase The intensity of the hybrid DNA will decrease relative to the light DNA, BUT WILL NEVER DISAPPEAR

In contrast to bacteria, eukaryotic chromosomes need multiple DNA replication origins because:

The linear nature of eukaryotic chromosomes and the large size of eukaryotic genomes necessitates multiple origins.

Strong promoter vs. weak promoter

The more the -10 and -35 resemble the consensus sequences, the stronger the promoter (RNAP binds readily and makes lots of sequences) - Gene expression is controlled, in part, by different sigma factors with diff. affinities for promoter DNA These things regulate bacterial transcription in different ways

Let's say your executing the experimental technique called DNA Foot Printing and you add RNA poly to your DNA. What can you infer if you see blurry yet distinct bands at the 10 and 35 sequences?

The promoter is weak and not much transcription is happening The promoter does not closely resemble the 10 & 35 consensus sequences

tRNAs interact with the ribosome only after

The small and large subunits have come together.

How do antibiotics work?

They target prokaryotic ribosomes (affect prokaryotic translation) but not eukaryotic ribosomes

Assume that a culture of E. coli was grown for approximately 50 generations in 15N (provided in the medium in the ammonium ion), which is a heavy isotope of nitrogen. You extract the DNA from the culture, and it has a density of 1.723 gm/cm3 (water = 1.00 gm/cm3). From the literature, you determine that DNA containing only the common form of nitrogen, 14N, has a density of 1.700 gm/cm3. Bacteria from the 15N culture were washed in buffer and transferred to 14N medium for one generation immediately after which the DNA was extracted and its density determined. Assuming that the molar percentage of adenine in the extracted DNA was 20 percent, what would be the expected molar percentages of the other nitrogenous bases in this DNA?

Thymine = 20%, Guanine = 30%, Cytosine = 30%

DNA polymerase I is thought to add nucleotides ____.

To the 3' end of DNA after RNA primer is removed

DNA polymerase III adds nucleotides ____.

To the 3' end of the primer

How to know which amino acid would aminoacylate a tRNA:

Transcribe codon in 5'->3' direction from anticodon (opposite direction of codon) Find codon in the table The CODON is what codes for the AA, NOT the anticodon

What would happen if the NTPs used for transcription had a hydrogen instead of a hydroxyl group?

Transcription wouldn't occur. No hydroxyl group to act as nucleophile an

Chain elongation - Translation

Transpeptidation: Peptide bond formation catalyzed by the 23S rRNA of the large subunit - Peptide at P site is transferred to A site = transpeptidation (A site amino acid nucleophilically attacks at the P site) - Peptidyl tRNA is now 1 AA longer - AA's are added to the C-terminus

True or False: DNA replication is discontinuous

True

True or False: In Eukaryotic cells, a distinct mRNA is produced for every one of the genes that is found on the molecule

True

True or False: Termination codons can be due to single base pair substitutions in a gene.

True

True or False: promoters are located upstream of the structural gene (portion of the gene that encodes a protein)

True

True/False: ssDNA must bind to ssRNA in the process of transcription.

True

True or False: the process of translation requires many protein factors

True Each phase requires unique translation factors: Initiation, elongation, and termination

True or False: transcription is more complex in eukaryotes than in prokaryotes

True (3 polymerases in euks, 1 in proks)

True or False: the Mg2+ at the DNA polymerase active site are essential

True! Act in Metal-ion catalysis of DNA polymerase to stabilize the neg. charged triphosphate and Asp at the active site

True/False: Bacteria carry out translation of mRNA and DNA replication in the same cellular space. True - both happen in the cytoplasm/cytosol. Translation - in ribosomes

True! Both happen in the cytosol/cytoplasm in bacteria (translation in ribosomes) NOT TRUE IN EUKARYOTES: DNA replication = nucleus, translation = cytoplasm/ribosomes

True or False: DNA Pol I and DNA Pol III both posses 5'->3' synthesis activity with an intrinsic error rate

True! Therefore, both require a 3'->5' exonuclease or proofreading function

True or false: More dense materials fall further in the centrifugation tube (15N)

True, 15N be closer to the bottom of the tube than 14N bc it contains the heavy isotope of Nitrogen

True or False: Transcription is the process of synthesizing a RNA polymer from individual NTPs in a DNA directed manner. This occurs with great fidelity

True. RNAP is DNA dependent

True or False: the catalytic site for dNMP incorporation is separate from the proofreading site

True. The active sites are adjacent to one another but separate so that DNA Pol can slide back and forth to position misaligned base in the 3'->5' exonuclease site and then slide forward to resume its synthesis activity

Some E. coli mutants contain defective DNA ligase. If the DNA produce by these bacteria were denatured and analyzed by gel electrophoresis, which of the following data would you see?

Two+ bands -- one band representing the leading strand of ssDNA with higher MW and one or more bands representing the lagging strand of ssDNA with lower MW Lagging strand has unsealed okazaki fragments so less new DNA than leading strand - lower MW

Which of the following mRNA codons would NOT be recognized by a tRNA that is charged with an amino acid?

UAA This is a stop codon which is recognized by release factors NOT tRNA

Gyrase (Topoisomerase)

Unwinds superhelical tension ahead of the replication fork as replication proceeds

3' OH end of DNA

Where new nucleotides are added (NO DNA synthesis on 5' end of DNA) 3' OH nucleophile attacks a phosphoryl group on dNTP - pyrophosphate side product (B and gamma phosphoryl groups) - a phosphoryl group incorporated into DNA

Is the direction of RNA synthesis the same as the direction of DNA synthesis?

Yes! Both 5'->3' synthesis

DNA replication requires _____ whereas transcription requires _____

dNTP NTP

E. coli DNA polymerase III:

is the principal DNA polymerase in chromosomal DNA replication.

The information gained from the DNA footprinting technique is the

location of a DNA binding protein on DNA.

Which of the following RNA molecules has the LEAST number of different sequences in a given organism?

rRNA

Which type or RNA makes up most of the RNA in our cells?

rRNA

The adaptor molecule in translation is

tRNA

Structure of tRNA

tRNA's are adaptors (connect mRNA to protein) and function to bring amino acids to the ribosome during translation tRNA possesses: - 5' Phosphate end - 3' CCA acceptor stem (where AA binds) - Anticodon region (interacts with triplet codon)