Midterm 3 - Transcription + translation

transcription and translation in bacteria and eukaryotes

Transcription

1. RNA is synthesized on DNA template

2. each triplet codon is complementary to anticodon of tRNA

Open reading frame

DNA sequence produces RNA with start + stop for transcription

• genes can overlap in bacteria and viruses

• single mRNA

Why is there more than one gene in each mRNA in bacteria and viruses?

The genome isnt as large so genes overlap

• one mRNA has multiple initiation points, different ORFs and more than one protein

RNA polymerase

synthesizes RNA using DNA template

• holoenzyme (all subunite together)

• no primer needed for initiation

What are the four steps of transcription?

1. promoter binding

2. initiation of txn

3. chain elongation

4. termination

promoter

It is a specific DNA sequence in the 5’ region upstream of txn initiation point

RNA pol binds at to this

• sigma subunit is responsible for promoter recognition

• E coli promoters have two promoter sequences

transcription start site

double helix is denatured to make template strand accessible for RNA polymerase

What must happen before elongation?

Removal of the sigma factor

chain elongation

occurs to synthesize RNA chains

• sigma subunits dissociate

• RNA polymerase can also proofread to replace mismatched nucleotide bases

Termination of Transcription

the 3’ end of mRNA forms a “hairpin” structure

• polyA (many A’s) have a weak interaction with the template DNA, causing RNA pol to stall → causes transcript to dissociate from the DNA

what is the sigma subunit of RNA Pol responsible for?

recognizing/binding to bacterial gene’s promoter

Name 6 differences in eukaryotic transcription

1. occurs in nucleus

2. mRNA must be “processed” before translation

3. chromatin must uncoil to make DNA accessible to RNA pol

4. RNA pol relies on transcription factors to scan/bind DNA

5. enhancers + silencers control transcription regulation

6. Termination of txn can be sequence-specific

Frameshift mutation

a result of insertion or deletion of a number of base pairs in a gene

What type of mutation helped lead to the understanding that genetic code is based on triplets?

frameshift

Polynucleotide phosphorylase

An enzyme used to artificially synthesize RNA templates.

• does not require a DNA template

How did scientists use RNA heteropolymers and polynucleotide phosphorylase?

used the heteropolymers to generate a synthetic message for the polynucleotide phosphorylase to synthesize mRNA with

• allowed them to determine the composition of triplet code words, but not the specific sequences

Triplet binding assay

radioactively charged tRNA, amino acid, and ribosomes were incubated together

• if radioactivity retained = the charged tRNA bound to the RNA triplet = can assign a specific codon

found that there is a reading frame and that there is a triplet code

What does it mean for transcriptional code to be degenerate?

all the amino acides are specified by 2-4 different codons

wobble hypothesis

aka the first two ribonucleotides in a codon are often more critical than the third

• The third base in an anticodon in tRNA can align in several ways = less spatially constrained = recognize more than one base in the codons of mRNA

Ordered genetic code

chemically similar amino acids often share 1-2 middle bases

• ex. AAA /AAG and AGA /AGG

nonsense mutation

A mutation that causes a codon to specify a stop codon, leading to premature termination

silent mutation

changes nucleotide but aa coded is the same

missense mutation

changes nucleotide and the aa coded

What are the three types of point mutations

1. silent

2. missense

3. nonsense

How many codons specify how many amino acids?

64 codons; 20 amino acids

how many codons specify for amino acids (excluding stop codons)

61

What does S mean in ribsomes

the measured rate of reaction - the particle’s density, mass, and shape

ribosome

consists of ribosomal proteins and rRNAs

• large and small subunit

the amino acid attaches to the __ end of tRNA

3’

tRNA is written 5’ to 3’. What is the anticodon for a 5’-GCC-3’?

GGC

monosome

when the two ribosomal subunits are assembled into a functional ribosome

polyribosome

a structure composed of 2+ ribosomes associated with an mRNA engaged in translation

• efficient use of components for protein synthesis

tertiary structure of a protein is dependent on the __ structure

primary

chaperone

protein that facilitates the folding of a polypeptide into its 3D shape of a functional protein

ubiquitin

a small protein that is covalently attached to misfolded proteins by __ ligases

• becomes a substrate for proteasome (protein complex with protease activity), which degrades the misfolded protein

What direction does the ribosome read mRNA?

5’ → 3’

What direction is protein synthesized?

N-terminus to C-terminus

RNA Pol I

transcribes rRNA

RNA Pol II

Transcribes protein coding genes (mRNA)

• highly regulated and dependent on various regulatory proteins

• enhancers, silencers

RNA Pol III

transcribes tRNA and rRNA for translation

What are the 3 components of post-transcriptional modifications to pre-mRNA?

1. add 5’ cap of modified guanine

2. add 3’ poly-A tail

3. remove introns

what is the purpose of the 5’ cap

• protects the 5’ end of mRNA

• helps with signaling → facilitates nuclear export and translation initiation

What is the purpose of the 3’ cap

prevents degradation and helps with nuclear export

• attached by poly-A polymerase

What is the purpose of introns

• alternative splicing

• transcriptional regulation

• gene evolution

Spliceosome

ribozyme that removes introns; in most eukaryotes

snRNAs are an essential part of it

• U1-U6

• Some of its RNA has catalytic activity

snRNAs

small nuclear RNAs

• complex with proteins to form snRNPs

snRNPs

complex of snRNAs and proteins

Unambiguous

each triplet specifies only one amino acid

degenerate

an amino acid can be specified by more than one triplet codon

What does it mean that the genetic code is nearly universal?

a single coding dirctionary is used by viruses, prokaryotes, archaea, and eukaryotes

What is an example of an exception to the universal genetic code?

Codon UGA usually codes for STOP, but in mitochondrial DNA in yeast and humans it codes for tryptophan

tRNA charging

tRNA molecules must be chemically linked to respective amino acids

• catalyzed by tRNA synthetase

tRNA synthetase

catalyzes aminoacylation

• 20 different synthetase (one for each amino acid)

  • Can bind to more than 1 tRNA because most aa.s have several codons that code for it

What the 4 types of R groups, and what biochemical properties do they have?

1. Nonpolar = Hydrophobic

2. Polar = Hydrophillic

3. Polar (+) = Acidic

4. Polar (-) = Basic

What type of bond connects amino acids?

peptide - holds amino + carboxyl group together

What components are required for initiation?

• small and large ribosomal subunits

• mRNA

• GTP

• charged initiator tRNA

• initiation factors (IF 1,2,3)

Aminoacyl site

Peptidyl site

Exit site

1st step of initiation

Initiation factors bind to small ribosome subunit, which binds to the Shine-Dalgarno mRNA sequence

Shine-Dalgarno sequence

initiates assembly of components required for initiation of translation in bacteria

• 5’-AGGAGG-3’

• is located before AUG

Kozak Sequence

increases efficiency of translation initiation

• [A/G][XX]AUG[G]

2nd and 3rd steps of initiation

1. IF2 stabilizes charged tRNA and mRNA into P site

2. large ribosomal subunit binds

3. requiers GTP

4. sets reading frame

Elongation

charged tRNAs are transported to the ribosome by elongation factors in to the A site

23s rRNA

catalyzes peptide bond formation between amino acid at A site and growing chain in P site

What is the purpose of the “rachet-like” action in the small ribosome subunit?

To “move” mRNA transcript to next site

Describes the process of termination of translation

1. signaled by stop codons, which do not specify any aa.

2. Release Factors bind to the A site and hydrolyzes polypeptide from p site tRNA

3. ribosomal complex breaks apart

What are two types of cis-regulators in transcription

1. enhancers - improves efficiency

2. silencers - reduces or blocks

What initially determines which DNA strand is the template strand + the direction RNA pol II moves along DNA?

The base sequence.

• Determines where the transcription factors bind in the promoter region.

What defines the exon/intron boundaries?

5’ GU and 3’ AG splice junction

Cis-acting elements

a sequence that regulates the expression of a gene located on the same chromosome

Trans-acting elements

a sequence that regulates the expression of a gene located on the homologous chromosome

Monocistronic mRNA

in eukaryotes

• has one open reading frame

• produces a single protein

Polycistronic mRNA

in prokaryotes

• has several open reading frames

• produces several proteins

operon

Clusters of genes and the regulatory region that controls the clusters

structural genes

genes coding for the primary structure of an enzyme

Catabolite-activating protein

diminishes expression of the lac operon when glucose is present

• form of catabolite repression

catabolite repression

selective inactivation of an operon by a metabolic product of the enzymes encoded by the operon

What is the relationship between glucose, cAMP, and lac?

glucose decreases levels of cAMP → represses transcription from lac operon

Poly-A binding proteins

bind to the Poly-A tail; bind to the 5’ cap binding proteins to form the loop

Describe the process of Closed Loop Translation

1. poly-A binding proteins bind to the 5’ cap-binding protein to form the loop

2. small and large ribosomal subunits come together to begin translation

What is the advantage of closed loop translation in eukaryotes?

efficient in ribosome recycling

Hemoglobin

tetrameric protein: 2 alpha + 2 beta subunit chains

• each subunit contains heme group that binds to oxygen

Sickle cell

Missense mutation for hemoglobin beta gene that causes changes to hemoglobin = less oxygen binding and delivery to tissues

How does missense mutation cause symptoms of sickle cell?

Polar amino acid (Glu) → nonpolar hydrophobic amino acid (Val) = does not want to face outside of cell so it faces inward = changes shape of RBC

What is the order of post-transcriptional processing?

1. addition of m⁷ cap to 5’ end

2. addition of poly-A cap to 3’ end

3. removal of introns

4. passage through nuclear membrane