Transcription and Translation

What carries out transcription? What are their properties? What must it recognize to start

1. RNA polymerase

2. do not require primer, lack 3’ to 5’ exonuclease activity

3. synthesize RNA 5’ to 3’

4. Must recognize start point for transcription and the template strand of DNA

what are the three factors in why we need transcription?

1. cell only has one DNA, which is not enough to go around

2. protect DNA from cytoplasmic environment

3. regulate the rate of protein synthesis:

   1. number of mRNA copies

   2. life-time of mRNA (degradation)

   3. RNA binding factors (translation)

Compare and contrast prokaryotic and eukaryotic RNA polymerases

• prokaryotic have a single RNA polymerase

  • transcribes all types of RNA

  • four subnits: a2bb

  • sigma factor binds to core enzyme and direct its binding to promotor (sigma 70)

• eukaryotic cell have 3 RNA polymerases

  • I = rRNA

  • II = mRNA

  • III = tRNA and 5s rRNA

  • Different polymerases recognize different type of promotoers

What is a gene and describe its structure

gene = segment of DNA containing information for the expression of RNA

contains:

• promoter: binding site for RNA polymerases and transcription factors

• promotor-proximal elements and enhancers: binding of nuclear receptors, co-activators, co-repressors

compare and contrast coding and template strnad

template: used by RNA polymerase during transcription; complimentary and ANTI PARALLEL to coding strand and RNA

CODING: analogous to produced RNA

discuss the promotor regions and the concensus sequences that are found to start transcription

e.coli: TATAAT - Pribnow box

Eukaryotic: TATA(A/T)A- TATA box

additional eukaryotic boxes: CAAT boxes, GC rich sequences, and enhancers

discuss the steps of bacterial transcription. What are DNA topoisomerase? compare/contrast RHO- independent and dependent termination signal

steps of transcription; uses holoenzyme RNA polymerase

1. recognize and bind to promoter

2. unwinds and separates DNA strands

3. sigma factor dissociates from RNA polymerase

4. RNA polymerase transcribs the DNA

DNA topoisomerase relaxes generated supercoils

Rho-independent: formation of hairpin loop by transcript results in release of RNA polymerase

RHO-dependent: binding of rho-factor causes release of polymerase

what is an operon? what is a cistron? What advantage does the lack of a nucleus provide for prokaryotes?

operon = where many protein-producing gene are linked together and controlled by a single promotor; usually proteins for same metabolic pathway

cistron = region of DNA that encodes for a single polypeptide

no nucleus = coupling of transcription and translation

what is rifampicin?

inhibits bacterial RNA polymerase and mitochondrial RNA polymerase

what are the five differences of between eukaryotic and bacterial transcription?

1. occurs in nucleus and in chromatin

2. more elaborate regulation

   1. more transcription factors and enhancers

   2. nucleosome-modifying enzymes (histone acetyltransferase)

   3. chromatin remodeling complexes

3. eukaryotes = 3 RNA polymerase

4. more elaborate mechanism of processing of transcripts

   1. modification of RNA transcript

   2. removal of introns

5. eukaryotic mRNA has information for only ONE polypeptide

what are GTFs? what are enhancers?

GTFs: general transcription factor

• interact with each other and RNA poly II to initiate transcription

enhancers: sequences that initiate transcription

• contain response elements: sequences that binds activators

• can bind transcription factors

• can be upstream or downstream

What are the modifications to the primary transcript of eukaryotic mRNA and its functions

has a 5’ cap (added during transcription)

• decreases rate of mRNA degradation

• serve as recognition site for binding to ribosome

poly -a - tail at 3’ end

• added by poly-a polymerase

• increases mRNA stability

has exons and introns

• exons = coding sequences

• introns are removed by splicing

describe the synthesis of eukaryotic RNA

1. by polymerase I

2. transcribed from a set of tandomly repeated genes

   1. about 1000 copies in genome

   2. rRNA is synthesized as a single large transcript

      1. cleaved into 18s, 28s, and 5.8s rRNAS

3. multiple Pol I can be on the same gene at a time

4. christmas tree appearance under electron microscope

5. forms the ribonucleoprotiein complex= ribosome

how are tRNAS produced?

1. cleavage of 5’ and 3’ ends

2. splicing to remove introns

3. replaces terminal 3’ terminal UU with CCA

4. multiple modification of bases for specificy of AA

what is Thalassemias?

• group of hereditary anemias; most common gene disorder in world

• affects synthesis of either alpha or beta chains of hemoglobin

• mutation result in a B+ phenotype in TATA box (A → G or A → C in the -28 to -31 region)

  • reduces accuracy of transcription start point

• other mutation futher upstream in promoter region (C→G at -87 and C→T at -88)

where do AA attach to at tRNA; how does it recognizes the mRNA codon?

at 3’ end of tRNA, via tRNA’s anticodon

describe the wobble’s hypothesis

explains why multiple codon code for one amino acid

Due to less precise base pairing between the first base of anticodon and 3rd base of codon

what are the four characteristic of the genetic code?

specificity: specific codon = always same AA

universality: exist at the dawn of evolution

redundancy: given AA may have more than one codon

nonoverlapping and commales: Code is read at the same starting spot and always 3 at a time

what are the different types of mutations

point mutations: one base is altered

1. silent: changed codon = same AA

2. missense: changed codon = different AA

3. nonsense: changed codon = become termination codon

Insertion, deletions, and frame-shift mutations

describe the mutation that causes sickle cell anemia and the result

missense mutation: GTG (valine) → GAG (Glu)

in each allele for B-globin

Results: oxygenated molecules are soluble but de-oxygenated = aggregate into insoluble fibers = deforms RBC into spiny or sickle-shaped cells

What are the components required for translation

1. template

   1. prokaryotic = polycistronic

   2. eukaryotic = monocistronic

2. ribosome

3. tRNAS

4. AA

5. ATP/GTP

   1. ATP = attach AA to tRNA

   2. GTP = binding of AA-tRNA to A site

   3. ATP and GTP are needed for initiation and termination steps

6. mRNA

describe the formation of aminoacetyl-tRNA

what are the three sites of the ribosome:

A: binds incoming aminoacyl-tRNA and specifies next AA to be added

P: occupied by peptidyltRNA; this tRNA carries the synthesized poly peptide

E: where empty tRNA exits

describe initiation of translation for prokaryotes and eukaryotes

prokaryotes:

• 16s RNA contain seq. complimentary to Shine-Dalgarno sequence of mRNA

  • binding of these two sequences on 30s

Eukaryotes:

• 40s binds to Cap structure of mRNA

describe the initiator tRNA

• recognizes AUG codon

• faciliated by IF-2 in e.coli and several IF in humans

• in pro. and mitochondria: initiator tRNA carries N-formylated methionine

• AA is attached on carboxyl end

  • catalyzed by peptidyltransferase

draw out the steps of translation

what does streptomycin do?

• binds to 30s subunit

• interferes with initiation of translation

• causes misreading of mRNA

• can cause perm. hearing loss

  • used mainly for treatment of TB

what does tetracycline do?

• binds to 30s subunit

• inhibits binding of AA-tRNA to A site

what does chloramphenicol do?

• interferes with peptidyltransferase of 50s Bacterial subunit

  • limited use in humans because also affects mitochondria

what does Ricin do?

• produced in seeds of castor oil plant

• N-glycosylase activity of ricin allows for cleavage of glycosidic bond within a large 60s subunit of eukaryotic ribosome

• this depurination = very rapidly and completely inactivates ribosome

list the three regulation of transcription

1. regulatory proteins: stabilize mRNA or can prevent binding of ribosome to mRNA

2. rare codons: rare codons have fewer tRNA in cell = slows down translation; usually located at beginning of mRNA

3. small interfering RNA = targets mRNA for degradation