Molecular Genetics (CH. 14-21)

Introns

Noncoding regions in RNA, common in eukaryotic genes but not prokaryotic

Exons

Coding regions of eukaryotic RNA

mRNA (messenger RNA)

Functions as a template/code for protein synthesis, carries genetic information from DNA into a ribosome.

Shine-Dalgaro Sequence

Consensus Sequence (AGGAGG) in prokaryotes that serves as the ribosome binding site in translation.

Codon

Three-nucleotide sequence on messenger RNA that codes for a single amino acid.

Start Codon (AUG)

Signifies the start of the protein-encoding sequence in mRNA

Stop Codons (UAA, UAG, UGA)

A nucleotide triplet within messenger RNA that signals a termination of translation

1. Addition of 5' cap
2. 3' cleavage and addition of poly(A) tail
3. RNA splicing

Post-Transcriptional Modifications of Pre-mRNA

1. Facilitates binding of ribosome to 5' end of mRNA
2. Increases mRNA stability
3. Enhances RNA splicing

3 functions of 5' cap

1. adds extra guanine nucleotide to 5' end of mRNA
2. adds methyl groups to guanine at 7 position to make methylguanosine
3. methyl groups are added to the 2' OH group of the first/second nucleotides adjacent to 7-methylguanosine

How is the 5' cap attached in pre-mRNA?

Protects mRNA from degradation, increases mRNA stability, and facilitates attachment of mRNA to ribosome. Helps export mRNA into the cytoplasm.

What is the function of the poly(A) tail?

1. 50-250 adenine nucleotides are added to the 3' end of mRNA
2. added after transcription by polyendylation
3. AAUAAA consensus sequence determines where cleavage takes place and after cleavage, the poly(A) tail is added.

How is the poly(A) tail created?

RNA splicing

Process by which the introns are removed from RNA transcripts and the remaining exons are joined together.

5' consensus sequence

GU A/G AGU: 5' splice site

3' consensus sequence

CAGG: 3' splice site

Branch Point

Adenine nucleotide in nuclear pre-mRNA introns that lies 18-40 nucleotides upstream of 3' splice site

Spliceosome

A large complex made up of proteins and RNA molecules that splices RNA by interacting with the ends of an RNA intron, releasing the intron and joining the two adjacent exons.

snRNPs (small nuclear ribonucleoproteins)

Complexes of proteins and small RNA molecules that function as components of spliceosomes during splicing (removal of introns from pre-mRNAs).

U1, U2, U4, U5, U6

What are the 5 snRNPs in a spliceosome?

1. the mRNA is cut at the 5' splice site
2. the 5' end of the intron attaches to branch point
3. a cut is made at 3' splice site
4. intron is released as a lariat
5. two exons are spliced together
6. the bond holding the lariat is broken and intron is degraded
7. spliced mRNA is exported to cytoplasm and translated

RNA splicing steps

1. alternative splicing
2. multiple 3' cleavage sites
3. RNA editing

Alternative Processing Pathways

Alternative Splicing

The same pre-mRNA can be spliced in more than one way to yield multiple mRNAs that are translated into different amino acid sequences and thus different proteins

Multiple 3' Cleavage Sites

Presence of more than one cleavage site for cleavage and polyendylation in pre-mRNA. Can change the 3' UTR and alters stability/translation.

Multiple mRNA's of different lengths

What does multiple 3' cleavage sites result in?

Polyadenylation

The addition of a poly-A tail at the 3' end of the mRNA.

RNA editing

Coding sequence of mRNA is altered after transcription, so the translated protein has a different amino acid than the one encoded by the gene.

Guide RNA (gRNA)

Contains partially complementary sequences to unedited mRNA and the two undergo base pairing. After the mRNA anchors to gRNA, the mRNA undergoes cleavage and nucleotides change according to the template provided by gRNA.

tRNA (transfer RNA)

RNA molecules that attaches to one type of amino acid and carries it to the ribosome, where it adds its amino acid to the growing polypeptide chain.

The tRNA has a cloverleaf shape with four arms, the acceptor arm forms a stem including 5' and 3' ends of tRNA.

What is the structure of tRNA?

Modified bases in tRNA that arise from chemical changes made to the original four bases after transcription.

What are ribothymidine and psedouridine?

Yes

Are all tRNA and rRNA molecules processed after transcription?

1. cleavage - large precursor tRNA cleaved to produce one tRNA molecule
2. splicing - introns removed
3. base addition - to 3' end
4. modification of bases produces mature tRNA, CCA sequence added to 3' end in some prokaryotes

tRNA processing steps

rRNA (ribosomal RNA)

Forms the primary component of ribosomes and helps export it into the cytoplasm, to assist in translation of mRNA. Makes up ~80% of cellular RNA.

1. methylation - methyl groups added to specific bases and to C-2' of ribose sugars
2. the RNA is cleaved into intermediates
3. ends are trimmed
4. the removal of introns by self splicing ribozymes

rRNA processing steps

Ribozymes

Catalytic RNA molecules that function as enzymes and can splice RNA

1. miRNA
2. siRNA
3. crRNA

What are the 3 small RNA's?

miRNA (micro RNA)

Regulate gene expression typically by blocking translation of selective mRNAs

siRNA (small interfering RNA)

Triggers degradation of other RNA molecules (only eukaryotes)

crRNA (CRISPR RNA)

Assists destruction of foreign DNA (only prokaryotes)

miRNA
origin: RNA transcribed from specific gene
cleavage of: single stranded RNA that forms short hairpins of double 3 RNA
size: 21-25 nucleotides
action: degradation of mRNA and inhibition of translation
target: genes other than those from which they were transcribed

siRNA
origin: mRNA, transposons, and viruses
cleavage of: RNA duplex or single stranded RNA that forms long hairpins
size: 21-25 nucleotides
action: degradation of mRNA
target: genes from which they were transcribed

Differences between miRNA and siRNA

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats, function in defense against foreign DNA

1. fragments of phage DNA are inserted into CRISPR array
2. array is transcribed into pre-mRNA
3. pre-crRNA cleaved, generating crRNA molecule
4. crRNA and cas9 protein combine to form effector complex
5. complex binds to foreign DNA and cleaves it

CRISPR - Cas9 system

1. Initiation
2. Elongation
3. Termination

What are the 3 steps in mRNA translation?

Wobble Hypothesis

Degeneracy is most common at the third position in a nucelotide triplet

Typically by having the same middle base in the amino acid sequence.

How is the genetic code ordered?

Degenerate Code

Multiple codons encode a single amino acid

1. non-polar, hydrophobic
2. polar, hydrophilic
3. charged (acid or base)

What are the 3 things that determine the characteristic of an amino acid

Peptide Bonds

How are amino acids joined?

Proteins

Made of chains of amino acids and form the largest organic component of the human body. They are essential not only to its fabric but also to its functioning.

1. Occurs on ribosomes
2. Requires mRNA, tRNA and rRNA
3. Requires helper proteins (IFs, EFs)
4. Requires energy (GTP)

Translation of mRNA = polymerization of amino acids into polypeptides: 4 key parts

1. mRNA typically more stable and longer-lived. - 7MG Cap and poly(A) tail play role
2. Differences in ribosomes, rRNAs, and proteins
3. More IFs and EFs needed.
4. Ribosome binding site on mRNA is different.
5. No fmet
6. Many ribosomes associated with RER.
7. Transcription and translation occur in different parts of the cell and are temporally separated.

Some differences in eukaryotic translation include:

1. Phosphorylation
2. Methylation
3. Acetylation
4. Addition of sugars
5. Cleavage of signal sequence

Post-translational modification examples in eukaryotes

Operon

Cluster of genes involved in the same process that are under the control of a single regulatory unit.

Allosteric

Changes shape when a small molecule (effector) binds to it.

Operon Domains

1. structural genes (typically encode enzymes)
2. regulatory unit (promoter and operator)
3. regulator gene (encodes DNA binding protein)

Active Form of Protein

Regulator protein is bound to DNA

Inactive Form of Protein

Regulator Protein is NOT bound to DNA

Negative Control of Transcription

Control when a regulator binds to DNA and shuts down transcription; repressor.

Positive Control of Transcription

Control when a regulator protein binds to DNA and transcription occurs; activator.

Regulator Protein

Protein produced by a regulator gene that binds to another DNA sequence and controls the transcription of one or more structural genes. Responsible for dictating positive or negative control.

Effector

Determines if the regulator protein is active (bound to DNA) or not. Establishes if the system is inducible or repressible.

Inducible Control

The effector is present and transcription occurs.

Repressible Control

The effector is present and transcription does NOT occur.

Lac Operon

Regulates lactose metabolism, under negative control (when the regulator protein binds to DNA it blocks transcription) and inducible (when the effector (lactose) is present is stimulates transcription).

lacI gene

Gene that codes for lac repressor, binds to lactose or operator DNA.

lacY gene

Gene that encodes permease and helps lactose enter the cell.

lacZ gene

Gene that codes for beta-galactosidase, which cleaves lactose into glucose and galactose.

Constitutive Genes

Genes that are expressed all the time

On

Is the Lac operon "on or off" when lactose (the effector) is present?

Off

Is the Lac operon "on or off" when lactose (the effector) is not present?

Partial Diploid

Bacterial cell that possesses two copies of some genes, one on the bacterial chromosome and the other on an extra piece of DNA (usually a plasmid); also called a merozygote.

Cis

Promoter / operator regions only act in _________ to the structural genes immediately adjacent; mutations in P / O region only affect adjacent nucleotides

lacI gene

Which gene is trans dominant?

Alter amino acid sequence of protein encoded by gene in which mutation occurs, affects lacZ and lacY.

What is the effect of structural-gene mutations and where are they found?

Affect transcription of structural genes on lacI, trans.

What is the effect of regulator-gene mutations and where are they found?

Affect transcription of structural genes on lacO, cis.

What is the effect of operator mutations and where are they found?

Affect transcription of structural genes on lacP, cis.

What is the effect of promoter mutations and where are they found?

Catabolite activating protein (CAP) binding to promoter

What is the element of positive control on the lac operon?

High

Do high or low levels of glucose slow down transcription of the lac operon?

cAMP-CAP complex

Binding of cAMP to CAP, increases rate of transcription.

trp Operon

Controls production of tryptophan (trp) and is under negative control (when the regulator protein binds to DNA it blocks transcription) and repressible (when the effector (trp) is present is inhibits transcription).

On

Is operon "on or off" when trp levels are low?

Two Mechanisms That Control Expression of trp Molecules

1. operator - repressor interaction
2. attenuation

Attenuation of trp Operon

Termination of transcription occurs with higher levels of Trp-tRNA prematurely

1. histone acetylation
2. chromatin remodeling
3. DNA methylation

What are the three ways chromatin structure is altered in eukaryotes?

Histone Acetylation

_________________________ of histone proteins by acetyltransferase proteins results in the destabilization of the nucleosome structure and increases transcription. The reverse reaction by deacetylases stabilizes nucleosome structure and decreases transcription.

Chromatin Remodeling

Allows for transcription to be initiated by increasing accessibility to the promoters by the transcription apparatus. Some of the remodeling complexes cause nucleosomes to move along the DNA, allowing DNA that was wrapped around the histone octamers to occupy a position between nucleosomes where it is more accessible to proteins involved in transcription initiation. Other complexes cause conformational changes in DNA, nucleosomes, or both to generate a more open configuration.

DNA methylation

Associated with decreased transcription. Methylated DNA sequences stimulate histone deacetylases to remove acetyl groups from the histone proteins, thus stabilizing the nucleosome and repressing transcription. Demethylation of DNA sequences is often followed by increased transcription, which may be related to the deacetylation of the histone proteins.

Core Promoter

General transcription factors and RNA polymerase bind to the _________________________________ to form the basal transcription apparatus.

Regulatory Promoter and enhancers

Transcriptional activator proteins bind to the ______________________________________ and to _______________________ to bring about normal levels of transcription.

Enhancers

DNA sequence that acts as a regulatory element to increase the level of transcription when regulatory proteins, such as transcription activators, bind to it.

Enhancers:
location: can be further away, can be up/downstream or within a gene.
orientation: orientation-independent
RNA: often transcribed
effect on transcription: efficient transcription
gene specific: no
function: time and tissue-specific gene expression

Promoters:
location: immediately adjacent and 5' to gene it controls
orientation: works in one direction
RNA: not transcribed
effect on transcription: basal levels of transcription
gene specific: yes
function: basic transcription

What distinguishes enhancers from promoters?

1. alteration of structures
2. transcription
3. mRNA processing
4. RNA stability
5. translation
6. post-translational modifications

6 levels of gene expression in eukaryotes

1. shrinking of poly(A) tail
2. removal of 5' cap
3. RNA binding proteins

What 3 things destabilize RNA?

RNA localization

Different RNA leaves nucleus and can be spread throughout the cell or concentrated on one side.

RNA silencing

Small RNAs inhibit gene expression in various ways, by cleavage of mRNA, inhibition of translation, or translational silencing.

Translational Control

Gene expression regulated by influencing the interaction of the mRNA transcripts with the ribosome

Post-Translational Modification

Chemical changes to the protein that may be critical to its final function after translation.

1. phosphorylation
2. acetylation
3. methylation
4. SUMOylation

Name 4 post-translational modifications of gene expression

General Transcription Factors

________________________________________________ stimulate and stabilize the basal transcription apparatus at the core promoter either directly through interaction with the basal transcription apparatus or indirectly through interaction with coactivator or with chromatin modifying proteins.