WEEK 1 (The central dogma) Flashcards
What is the Central dogma of molecular biology?
An explanation of the flow of genetic information within a biological system
What is an essential part of the machinery for synthesising proteins?
RNA-based enzymatic catalysis
What is the “RNA world hypothesis” and what does it propose?
The ability of DNA and RNA to base pair with free nucleotides suggests a scenario in which an RNA sequence spontaneously formed that was then able to self-replicate, ultimately giving rise to many self-propagating copies of itself
This proposes that life began as RNA
What was the importance of Archibald Garrod?
- Provided the first meaningful insight into gene function
- Discovered the relationship between a genetic defect, a specific enzyme and a specific metabolic condition
- One disease investigated was ALCAPTONURIA
Describe Alcaptonuria
A condition where urine becomes dark on exposure to air. Individuals with Alcaptonuria lack an enzyme in their blood that OXIDISED HOMOGENTISIC ACID (a compound formed during the breakdown of PHENYLALANINE and TYROSINE). As homogentisic acid accumulates, it is secreted in the urine and darkens in colour when oxidised in air.
What is the function of Retroviruses?
Retro viruses carry RNA as their genetic information. When they invade their host cell they convert their RNA into a DNA copy using REVERSE TRANSCRIPTASE.
This modifies the central dogma
DNA <-> RNA <-> PROTEIN
Most protein-coding genes occur only once in the genome and are called ____________________
Single-copy genes
What are exons and introns?
Exons = The protein-coding regions of the split genes
Introns = Intervening sequences spliced out before the mRNA leaves the nucleus
What are the four steps for Protein synthesis?
1) Transcription
2) RNA processing
3) Translation
4) Post-translation processing
What is Transcription/RNA synthesis?
The process whereby the information held in the nucleotide sequence of DNA is transferred to RNA
What are genes encoding mRNAs called?
Protein-coding genes
When is a gene said to be expressed?
When its genetic information is transferred to mRNA and then to protein
What are the properties of primary transcripts?
- Do not exist within the cell as naked RNA but become associated with proteins
- Fleeting existence
- Processed into smaller, functional RNAs by a series of “cut and paste” reactions
What is the Primary transcript/Pre-RNA?
The initial precursor RNA equivalent in length to the full length of DNA transcribed
What is the Transcription unit?
The corresponding segment of DNA from which a primary transcript is transcribed
What are the three major types of eukaryotic RNAs?
- Messenger RNA (mRNA) = provides the PLAN for the polypeptide chain
- Ribosomal RNA (rRNA) = provides the PLATFORM for protein synthesis
- Transfer RNA (tRNA) = TRANSLATES the message on the mRNA into a polypeptide chain
Transcription
- Uses RNA polymerase
- Proceeds in the same direction as replication (5’ to 3’)
- Forms a complementary strand of mRNA
- It begins at a PROMOTOR SITE which signals the beginning of a gene
- After end of gene there is a TERMINATOR SEQUENCE that tells RNA polymerase to stop transcribing
In any gene how many DNA strands acts as a template for transcription?
One
What does the sequence of nucleotides in RNA depend on?
The nucleotide sequence in the DNA template
Which enzyme is used to transcribe DNA into RNA?
RNA Polymerase
What is the role of RNA Polymerase in Transcription?
- Recognises the beginning of the gene to be transcribed
- Catalyse the formation of phosphodiester bonds between the nucleotides that have been selected according to the sequence within the DNA template
What is the functions of DNA-dependent RNA Polymerases?
- Search DNA for initiation site
- Unwinds a short stretch of double helical DNA to produce a single-stranded DNA template
- Selects the correct ribonucleotide and catalyses the formation of a phosphodiester bond
- Detects termination signals where transcript ends
What is the Promoter?
The site on the DNA to which an RNA Polymerase molecule binds prior to initiating transcription
What is the importance of Transcription factors?
Helps cellular RNA Polymerases to recognise promoters since they cannot on their own
What is the significance of the Promoter?
- Provides a binding site for Polymerase
- Contains the information that determines which of the two DNA strands is transcribe and the site at which transcription begins
What is a Transcription bubble?
A molecular structure formed during DNA transcription when a limited portion of the DNA double strand is unwound
Describe what happens after RNA Polymerase binds to the Promoter
1) RNA Polymerase moves along the template DNA strand towards its 5’ end ( 3’ to 5’ direction)
2) As the Polymerase progresses, the DNA is temporarily unwound
3) Polymerase assembles a complementary strand of RNA that grows starting from its 5’ terminus in a 3’ direction
How many base pairs of DNA does a Polymerase cover, the Transcription bubble compose of and the segment present in a DNA-RNA hybrid include?
- Polymerase covers approximately 35 BASE PAIRS of DNA
- Transcription bubble composed of 15 BASE PAIRS
- Segment present in a DNA-RNA hybrid includes 9 BASE PAIRS
What is the difference between RNA Polymerase I, II, III, IV & V and the RNAs they synthesise?
ENZYME: RNA POLYMERASE I
RNAs SYNTHESISED: Larger RNAs
ENZYME: RNA POLYMERASE II
RNAs SYNTHESISED: mRNAs, small nuclear RNAs & Telomerase RNA
ENZYME: RNA POLYMERASE III
RNAs SYNTHESISED: tRNAs
ENZYME: RNA POLYMERASE IV, V (Plants only)
RNAs SYNTHESISED: SiRNAs
Which enzyme transcribes the genes that code for most of the ribosomal RNAs?
RNA Polymerase I
Which enzyme synthesised all messenger RNAs?
RNA Polymerase II
What is the same in Eukaryotes and Bacteria?
The chemical reaction catalysed by RNA POLYMERASE I, II & III which is the formation of phosphodiester bonds between nucleotides
What are the properties of RNA POLYMERASE I?
- Located in the NUCLEOLUS of the cell
- Specialised nuclear substructure where rRNA is synthesised by transcription and assembled into ribosomes
- rRNA are ribosome components & are important in TRANSLATION
What are the properties of RNA POLYMERASE II?
- Located in the NUCLEUS
- Contain transcription factors and transcriptional regulators
- Synthesises all proteins that code for the nuclear pre-mRNAs in eukaryotic cells
- Responsible for transcribing most of the eukaryotic genes & found in human genes
What are the properties of RNA Polymerase III?
- Located in the nucleus
- 14+ distinct subunits
- Transcribes transfer RNA (tRNA), ribosomal RNA (rRNA) and other small RNAs
- Some of its target points are important for the normal functioning of the cell
Describe what RNA Polymerase looks like
A molecule shaped like a crab claw enclosing a positively charged internal channel
What are the differences between Prokaryotic RNA Polymerase and Eukaryotic RNA Polymerase?
PROKARYOTIC RNA POLYMERASE
- Consists of Four subunits & a sigma factor
- Only one single type
- Core enzyme contains FIVE subunits
- SIZE: 400 kDA
- Synthesises polycistronic RNA
- Prokaryotes regulate transcription with the use of different types of sigma factors
EUKARYOTIC RNA POLYMERASE
- FIVE types
- Contains 10-20 subunits
- SIZE: 500 kDA
- Synthesises monocistronic RNA
- Transcription is regulated by the presence of different types of RNA Polymerases
What are the three phases of transcription?
1) Initiation
2) Elongation
3) Termination
What are the start and stop codons?
- START: AUG (Methionine)
[sometimes GUG is used during INITIATION but GUG normally encodes valine] - STOP: UAG, UAA, UGA
Describe the RNA Polymerase that transcribes E. coli genes
RNA Polymerase is made up of FIVE SUBUNITS (α, α, β, β’ & σ)
What is the role of the σ factor?
To recognise the PROMOTER which lies just upstream of the gene to be transcribed
Describe the two important regions that promoters contain
- PRIBNOW BOX/-10 BOX is centred around nucleotide -10 and has the sequence TATATT
- -35 BOX is centred near nucleotide -35 and has the sequence TTGACA
What is RNA synthesis facilitated by?
General transcription factors
What are hnRNAs (heterogenous nuclear RNAs)?
Precursors to smaller cytoplasmic mRNAs
Describe what happens after the σ binds to the promoter sequence
1) The σ brings the other subunits (two of α plus one each of β and β’) of RNA Polymerase in contact with the DNA to be transcribed forming the CLOSED PROMOTER COMPLEX
2) Two strands of DNA separates enabling one strand to act as a template for the synthesis of an RNA molecules forming the OPEN PROMOTER COMPLEX
3) Separation is helped by the AT-rich sequence of the -10 box (relatively easy to separate strands since adenine and thymine only have two hydrogen bonds between them)
4) DNA unwinds and rewinds as RNA Polymerase advances along the double helix, synthesising an RNA chain as it goes producing a TRANSCRIPTION BUBBLE
Describe what happens after the σ binds to the promoter sequence
1) The σ brings the other subunits (two of α plus one each of β and β’) of RNA Polymerase in contact with the DNA to be transcribed forming the CLOSED PROMOTER COMPLEX
2) Two strands of DNA separates enabling one strand to act as a template for the synthesis of an RNA molecules forming the OPEN PROMOTER COMPLEX
3) Separation is helped by the AT-rich sequence of the -10 box (relatively easy to separate strands since adenine and thymine only have two hydrogen bonds between them)
4) DNA unwinds and rewinds as RNA Polymerase advances along the double helix, synthesising an RNA chain as it goes producing a TRANSCRIPTION BUBBLE
When is the σ factor released?
When the RNA chain is about 10 bases long
Describe what happens after the σ factor is released
1) β-subunit of RNA Polymerase binds ribonucleotides and joins them together by catalysing the formation of phosphodiester bonds as it moves along the DNA template
2) β’-subunit helps to keep the RNA polymerase attached to DNA & the two α-subunits help RNA Polymerase to assemble on the promoter
3) Terminators are reached which cause RNA Polymerase to stop transcribing DNA
What is rho?
A protein that recognises terminator sites and frees the RNA from the DNA
[terminates by a process called RHO-DEPENDENT TERMINATION]
What does a terminator sequence consist of?
Two regions rich in Guanine and Cytosine that are separated by 10 base pairs
Describe how Transcription in Eukaryotes differs from that in Prokaryotes
- EUKARYOTES: occurs in NUCLEUS
- EUKARYOTES: requires TRANSCRIPTION FACTORS (binding of Polymerase to DNA template, initiation of transcription, elongation & termination)
- EUKARYOTES: requires compact CHROMATIN FIBER characterised by nucleosome coiling - to be uncoiled & DNA made accessible to RNA polymerase and other regulatory proteins
- EUKARYOTES: Initiation and regulation of transcription entail a more extensive interaction between cis-acting DNA sequences and trans-acting protein factors involved in stimulating and initiating transcription
What is Processing?
Alteration of the primary RNA transcript to produce mature eukaryotic mRNA involving many complex stages
What are Transcription factors?
Proteins that bind to DNA near the start of transcription of a gene and either inhibit or assist RNA polymerase in initiation and maintenance of transcription
What are the two categories of transcription factors?
- General Transcription Factors (GTFs)
- Transcriptional activators and repressors
What are the properties of mRNAs?
- Contain a continuous sequence of nucleotides encoding a specific polypeptide
- Found in the cytoplasm
- Attached to ribosomes when they are translated
- Contain a significant noncoding segment
- EUKARYOTIC mRNAs have special modifications at their 5’ and 3’ ends that aren’t found on bacterial mRNAs, tRNAs or rRNAs
[3’ end of nearly all eukaryotic mRNAs has a string of 50-250 adenosine residues that form a poly (A) tail whereas the 5’ end has a methylated guanosine cap]
What is the function of noncoding segments of RNA?
Contain sequences with important regulatory roles
Describe the structure of the human β ‐globin mRNA
mRNA contains
- 5’ methylguanosine cap
- 5’ and 3’ noncoding region that flanks the coding segment
- 3’ poly (A) tail
[length of poly(A) tail is variable]
What are the stages of mRNA processing?
1) Capping
2) Addition of poly A tail
3) Splicing
What is the reason behind capping and addition of Poly A tail?
RNA is an unstable molecule and the capping of eukaryotic mRNA at their 5’ ends and the addition of the poly-A tail to their 3’ ends increases the lifetime of mRNA molecules by protecting them from digestion by nucleases
Describe Capping and Addition of Poly A tail
1) 5’-terminal triphosphate group is modified by the addition of GUANOSINE via a 5’-5’-phosphodiester link. The guanosine is METHYLATED to form the 7-METHYL GUANOSINE CAP
2) 3’ ends are modified by the addition of a long stretch of ADENOSINE RESIDUES, the POLY-A TAIL
Where does removal of introns take place?
Within the nucleus
Describe one of the rules in splicing
The first two bases following an exon are always GU and the last two bases of the intron are AG
Which types of RNA are involved in splicing?
small nuclear RNAs (snRNAs)
[these are complexed with a number of proteins to form a SPLICEOSOME]
Describe what happens in splicing
1) One of the snRNAs is complementary in sequence to either end of the intron sequence
2) Binding of this snRNA to the intron by complementary base pairing brings the two exon sequences together which causes the intron to loop out
3) Proteins in the SPLICEOSOME remove the intron and join the exons together
What is alternative splicing?
A process in which exons are removed which allows the same gene to give rise to different proteins at different times or in different cells
What is the reason for Alternative splicing?
A way to get more than one protein product out of the same gene and a way to control gene expression in cells
[very common in higher eukaryotes]
Describe Mitochondrial DNA
- Circular
- One heavy strand and one light strand
- Most of the coding nucleotides are found on the heavy strand
- Two promoters therefore both strands of mitochondrial DNA are used for transcription
